Synthetic hydrogel composite

ABSTRACT

Cellulose-reinforced hydrogels may include a cellulose nanofiber network and an interstitial hydrogel portion within interstitial regions of the cellulose nanofiber network, the interstitial hydrogel portion comprising polyvinyl alcohol (PVA), wherein the hydrogel component has a crystallinity of 20% or greater.

CLAIM OF PRIORITY

This patent application is a divisional of U.S. patent application Ser.No. 17/845,881, filed Jun. 21, 2022, titled “SYNTHETIC HYDROGELCOMPOSITE,” which claims priority to U.S. Provisional Patent ApplicationNo. 63/338,439, filed on May 4, 2022, titled “SYNTHETIC HYDROGELCOMPOSITE,” each of which is herein incorporated by reference in itsentirety.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

BACKGROUND

Every year, approximately 900,000 people in the United States sufferfrom damage to the articular cartilage that lines the ends of the bones.Osteoarthritis (OA) is an example of a degenerative joint disease thatis a common cause of disability. Articular cartilage lesions mostcommonly occur in the knee and can cause debilitating pain. Cartilagelacks blood vessels and has a very limited capacity for healing. Oneapproach to treating cartilage lesions is to attempt to regrow cartilagewith a technique such as microfracture or autologous chondrocyteimplantation. Unfortunately, these methods have high failure rates,prolonged rehabilitation times, and show decreasing efficacy in olderpatients. Implantation of fresh osteochondral allografts can speedrecovery as they eliminate the need to regrow cartilage and.Unfortunately, the small supply of fresh allografts limits the number ofthese procedures. Failure of these treatment strategies usually leads tomore invasive total knee replacement. While total knee replacement maybe successful in older patients, it may not be suitable for youngerpatients for whom the implant is likely to fail within their lifetime,thus requiring a second invasive surgery. Thus, there is a clear needfor minimally invasive treatment options that treat cartilage lesionswith a low failure rate, enable rapid recovery, and are widelyavailable.

Given the need for a less invasive alternative to total knee replacementfor treatment of OA, there are ongoing efforts to replace damagedcartilage with a device made of traditional orthopedic materials, suchas a cobalt-chrome alloy or ultra-high-molecular-weight polyethylene.However, these materials have a much higher coefficient of friction(COF) than cartilage and may cause an unacceptable level of wear on theopposing cartilage surface. In addition, these materials are muchstiffer than cartilage and may therefore cause an abnormal stressdistribution in the joint, potentially contributing to the damage ofsurrounding cartilage.

Hydrogels, polymer networks swollen with water, are a promisingsynthetic material for replacement of cartilage because hydrogels can bemade to have similar mechanical and tribological properties as naturalcartilage. However, there is a need to improve the physical propertieshydrogels to withstand the wear and tear that an implant may encounter.For example, there is a need for hydrogels to be at the higher end ofthe range of strengths reported for cartilage while having a similarmodulus, coefficient of friction, and resistance to wear as cartilage.Described herein are methods, hydrogel compositions, and apparatuses(e.g., implants) that may address these needs.

SUMMARY OF THE DISCLOSURE

This disclosure relates generally to artificial cartilage materials inimplants suitable for repair of cartilage, including hydrogel compositesand methods and for attaching a hydrogel composite to a surface of animplant.

Described herein are hydrogel materials for use as artificial cartilagein implants. A hydrogel may be infused in a nanofibrous material (e.g.,a nanofiber network) and bound to a surface of an implant, such as aporous base. The composite hydrogel has physical properties, such asstrength, modulus and wear resistance, and coefficient of friction (COF)that approximates or exceeds that of healthy cartilage bound to bone.The methods may involve a strengthening process to increase thecrystallinity and decrease the water content of the hydrogel, therebyimproving its mechanical properties for implementation as cartilagereplacement. As used described herein, strengthening of a hydrogel mayinclude one or more steps of drying, annealing, and rehydrating toinfluence the crystalline structure of the hydrogel. The methods mayfurther involve securing a nanofibrous material to a surface of animplant, infiltrating a hydrogel into the nanofiber network, andannealing the hydrogel.

Approaches to creating synthetic cartilage by infiltrating a hydrogelinto a nanofiber network for mimicking cartilage are described inInternational Patent Application No. PCT/US2021/040031, which isincorporated herein by reference in its entirety. The methods describedherein may be used to form hydrogels that match or exceed the higher endof the range of strength of cartilage, while having a similar modulus,coefficient of friction, and resistance to wear of cartilage.

Described herein are hydrogels and methods of making and using them formimicking or replacing cartilage, and that may be interdigitated with ananofibrous network, such as a cellulose nanofiber network. Theincorporated hydrogel may have a crystalline structure that imparts hightensile and/or compressive strength to the hydrogel. In some examples, areinforced hydrogel for use in an implant described herein may include across-linked cellulose nanofiber network; and a hydrogel infused withininterstitial regions of the cross-linked cellulose nanofiber network,wherein the hydrogel has a crystallinity of 20% or greater. In someexamples, the hydrogel comprises polyvinyl alcohol (PVA). In any ofthese examples the hydrogel may exclude (or substantially exclude)PAMPS. The hydrogel may be >90% PVA(e.g., >92%, >93%, >94%, >95%, >96%, >97%, >98%>99%, etc.) of PVA thathas been annealed as described herein.

As demonstrated herein, the crystallites formed during annealingstrengthens an otherwise amorphous polymer hydrogel by acting ascross-links that redistribute applied stresses and hinder crackpropagation. The crystallites also increase the solid content andstrength of the hydrogel by reducing the amount of water taken up by thePVA after annealing.

Described herein are implants comprising: an implant body and acellulose-reinforced hydrogel material comprising: a cross-linkedcellulose nanofiber network bonded to the porous surface of the implantbody by a cement; and a hydrogel impregnated in the cross-linkedcellulose nanofiber network, wherein the hydrogel has a crystallinity of20% or greater. The implant body may include a porous surface. Forexample, the implant body may be a titanium body with a bone-facingporous surface and a hydrogel-facing non-porous surface.

The hydrogels described herein may have a water content conducive toimparting high tensile and/or compressive strength to the hydrogel. Insome examples, the hydrogel may have at least 20% by weight (wt %) ofwater and have a tensile strength exceeding that of cartilage, e.g.,exceeding 40 Megapascals (MPa).

The composition of the interstitial hydrogel may be chosen to maximizecrystallinity. For example, some hydrogel polymers and/or polymermixtures have been found to hinder the crystalline formation, therebydecreasing the tensile and compressive strength of the compositehydrogel.

In general, the hydrogels may be comprised of one or more polymers thatare conducive to forming crystalline structures. In some examples, thehydrogel may include polyvinyl alcohol (PVA). In some cases, thehydrogel may include only one type of polymer. In some variations, thehydrogel is comprises of one or more of: polyvinyl alcohol (PVA),poly(2-acrylamido-2-methyl-1-propanesulfonic acid sodium salt (PAMPS),poly-(N,N′-dimethyl acrylamide) (PDMAAm), copolymers of 1-vinylimidazoleand methacrylic acid, amphiphilic triblock copolymers, polyampholytehydrogels, a PVA-tannic acid hydrogel, a poly(N-acryloyl) glycinamidehydrogel, polyacrylic acid-acrylamide-C18 hydrogel, Guanine-boric acidreinforced PDMAAm, polyelectrolyte hydrogels, apoly(acrylonitrile-co-1-vinylimidazole) hydrogel (e.g., a mineralizedpoly(acrylonitrile-co-1-vinylimidazole) hydrogel), a polyacrylicacid-Fe3+-chitosan hydrogel, a poly(methacrylic acid) gel, a Grapheneoxide/Xonotlite reinforced polyacrylamide (PAAm) gel, a poly(stearylmethacrylate)-polyacrylic acid gel, an annealed PVA-polyacrylic acidhydrogel, supramolecular hydrogels from multiurea linkage segmentedcopolymers, polyacrylonitrile-PAAm hydrogel, a microsilica reinforcedDMA gel, a Agar-polyhydroxyethylmethacrylate gel, apolyfacryloyloethyltrimethylammonium chloride hydrogel, apoly(3-(methylacryloylamino)propyl-trimethylammonium chloride hydrogel,a poly(sodium p-styrenbesulfonate) hydrogel, a polyethylene glycoldiacrylate hydrogel, and a polyethylene glycol hydrogel. In some casesit may be beneficial to exclude PAMPS (e.g., having no PAMPS, havingless than 0.1%, less than 0.5%, less than 1%, etc.).

The nanofiber network may comprise a cellulose nanofiber network. Thenanofiber network may comprise a cross-linked cellulose nanofibernetwork. In some examples the nanofiber network comprises a bacterialcellulous (BC). Additionally or alternatively, the nanofiber network maycomprise at least one of: electrospun polymer nanofibers, poly(vinylalcohol) (PVA) nanofibers, aramid nanofibers, aramid-PVA nanofibers,wet-spun silk protein nanofiber, chemically crosslinked cellulosenanofiber, and polycaprolactone (PCL) fibers.

A cellulose-reinforced hydrogel may include: a cellulose nanofibernetwork; and a hydrogel impregnated in the cellulose nanofiber network,wherein the hydrogel has a crystallinity of 20% or greater. Thecellulose-reinforced hydrogel material may have a tensile strength of 40MPa or greater. The cellulose-reinforced hydrogel material may have acompressive strength of 59 MPa or greater.

Described herein is a cellulose-reinforced hydrogel comprising a watercontent of at least 20 wt % and a compressive strength exceeding 59 MPa.The cellulose-reinforced hydrogel may comprise bacterial celluloseand/or a hydrogel comprising polyvinyl alcohol (PVA).

Described herein is a method of forming a cellulose-reinforced hydrogelcomprising: infiltrating a hydrogel in a cellulose nanofiber network toform the cellulose-reinforced hydrogel; and annealing the hydrogel toincrease a crystalline content of the hydrogel. Annealing the hydrogelmay include heating the cellulose-reinforced hydrogel. Annealing thehydrogel may include heating the cellulose-reinforced hydrogel todecrease a water content of the hydrogel. In some examples, thecellulose-reinforced hydrogel may be heated to a temperature rangingfrom 90-140° C. Annealing the hydrogel may include rehydrating thehydrogel. Rehydrating the hydrogel may include increasing a watercontent of the hydrogel to at least 20 wt %. The method may furtherinclude removing excess hydrogel from a surface of the cellulosenanofiber network. Removing excess hydrogel may include removing theexcess hydrogel by hand or by molding the cellulose-reinforced hydrogel.

Described herein is an implant knee resurfacing comprising: a topbearing surface comprising a cellulose-reinforced hydrogel comprising: acellulose nanofiber network; and a hydrogel impregnated in the cellulosenanofiber network, wherein the hydrogel has a crystallinity of 20% orgreater.

The hydrogel (e.g., cellulose-reinforced hydrogel) may be attached to ametallic base with a shear strength exceeding 0.2 MPa.

When used for partial knee resurfacing, the implant may be configured towear an opposing cartilage surface to an extent not significantlygreater than the extent to which cartilage wears cartilage. A topbearing surface of the implant may have a coefficient of friction (COF)that is not statistically different from that of cartilage.

The implants described herein may be configured as a medical implant,and may include a tissue engaging portion (e.g., a bone engaging portionsuch as a rod, screen, nail, etc.). A first surface of the implant, towhich a nanofiber network may be secured, may be porous. For example,the first surface may be greater than 40% porous to a depth of 1 mm orgreater.

The nanofiber network may be secured to the implant (e.g., to a poroussurface of the implant) by any appropriate method. For example, thenanofiber network may be secured to the implant by a cement, such as anα-TCP cement. In some examples the cement comprises one or more of: zincoxide eugenol, glass ionomer, calcium silicate, polycarboxylate cement,zinc phosphate, resin-based (dental) cements, such as acrylate ormethacrylate resin cements, which may contain silicate or other types offillers in an organic resin matrix (for example, a methacrylate cementsuch as “RelyX™ Unicem 2 Self-Adhesive Resin Cement,” or “RelyX™Ultimate Adhesive Resin Cement”), and resin-modified glass ionomercement. The cement may include an adhesive, such as (but not limited to)phosphoserine (PPS). In some variations the cement may include particlesfor reinforcement, such as stainless steel particles (e.g., stainlesssteel powder, SSP).

The cement may extend at least 5 microns into the nanofiber network fromthe first surface (e.g., 6 microns or more, 7 microns or more, 8 micronsor more 10 microns or more, 15 microns or more, 20 microns or more,etc.). The cement may not be bonded to the hydrogel. In some examples,the cementing may be completed (and the cement set or dry) beforeimpregnating with the hydrogel.

The cement may be bonded to the nanofiber network but not be bonded tothe hydrogel directly. This may be a consequence of the method offorming the network-reinforced hydrogel, in which the nanofiber network(e.g., the cellulose nanofiber network) is first secured (e.g.,cemented) to the implant body, before impregnating the hydrogel. Thecement may be cured onto the nanofiber network so that it does notdirectly bond to the hydrogel.

Other adhesives may include surgical adhesives such as cyanoacrylate,gelating/resorcinol/formaldehyde (GRF), and/or fibrin.

The implant may be formed of any appropriate biocompatible material. Forexample, the surface of the implant body may be titanium. The surface ofthe implant body may be one or more of: a stainless steel alloy, atitanium alloy, a Co—Cr alloy, tantalum, gold, niobium, bone, Al oxide,Zr oxide, hydroxyapatite, Tricalcium phosphate, calcium sodiumphosphosilicate, poly(methyl methacrylate), polyether ether ketone,polyethylene, polyamide, polyurethane, or polytetrafluoroethylene.

As mentioned, the attachment surface in which a nanofiber network issecured may be porous. Alternatively, the attachment surface may benon-porous. For example, the attachment surface may be 20% or greater(30% or greater, 40% or greater, 50% or greater, 60% or greater, 70% orgreater, etc.) porous, to a depth of 0.5 mm or greater (e.g., 0.6 mm,0.7 mm, 0.8 mm, 0.9 mm, 1 mm or greater, etc.). As used herein, thepercentage that the surface if porous (e.g., the percent porosity of thesurface) may refer to the percentage of the surface within the depththat is absent, forming open spaces within the surface. These openspaces may refer to pores, some of which may be connected (e.g., influid connection) with each other. The attachment surface may generallybe referred to herein as a top bearing surface because it may beconfigured to contact other surfaces (e.g., bone surfaces, etc.), andmay be added to a load-bearing surface.

In any of these apparatuses (e.g., devices, systems, includingimplants), at least a portion of the nanofiber network may bemineralized. For example, at least a portion, such as the region nearthe interface with the surface, may be mineralized with hydroxyapatite.The mineralization may extend at least 5 microns into the nanofibernetwork (e.g., at least 7 microns, at least 8 microns, at least 9microns, at least 10 microns, at least 15 microns, at least 20 microns,etc.) from the surface.

In general, the nanofiber network may be coupled to the top bearingsurface of the implant. The cross-linked cellulose nanofiber network maybe attached over the top load surface by clamping and/or by adhesive.For example, the nanofiber network may be bonded by cement to the topload surface; in some examples, the cement is not bonded to thehydrogel; the cement is only bonded to the nanofiber network.Alternatively, in some examples the nanofiber networks may be coupled tothe implant, so that the nanofiber network, is secured over the topbearing surface without the use of a chemical adhesive, such as anepoxy. Instead, the nanofiber network may be secured over the topbearing surface by a clamp. For example, a clamp may secure thenanofiber network (e.g., one or more sheets of BC) over the top bearingsurface around a periphery of the top bearing surface. Thus, in general,the use of an adhesive (such an epoxy) is optional.

Any appropriate implant may be used. The surface of the implant (e.g.,top bearing surface, which may be equivalently referred to as simply thebearing surface) may be at least at the region to which the nanofibernetwork is attached over, may be titanium, stainless steel, etc. thebearing surface (e.g., top bearing surface) may be convex, flat,concave, or some mixture of these. For example, the surface of theimplant body may comprise one or more of: a stainless steel alloy, atitanium alloy, a Co—Cr alloy, tantalum, gold, niobium, bone, Al oxide,Zr oxide, hydroxyapatite, Tricalcium phosphate, calcium sodiumphosphosilicate, poly(methyl methacrylate), polyether ether ketone,polyethylene, polyamide, polyurethane, or polytetrafluoroethylene.

Also described herein are methods of making and/or using these implants.For example, described herein are methods of attaching a hydrogel to asurface so that the hydrogel is secured to the surface with a shearstrength of greater than 1 MPa. Any of these methods may include:infiltrating a hydrogel in a cellulose nanofiber network to form thecellulose-reinforced hydrogel; and annealing the hydrogel to increase acrystalline content of the hydrogel. For example, annealing the hydrogelmay include heating the cellulose-reinforced hydrogel. In some examples,annealing the hydrogel may include heating the cellulose-reinforcedhydrogel to decrease a water content of the hydrogel. For example, thecellulose-reinforced hydrogel may be heated to a temperature rangingfrom 90-140° C. In some cases, annealing the hydrogel may includerehydrating the hydrogel. Rehydrating the hydrogel may includeincreasing a water content of the hydrogel to at least 20 wt %. Themethods may also include removing excess hydrogel from a surface of thecellulose nanofiber network. Removing excess hydrogel may includeremoving the excess hydrogel by hand or by molding thecellulose-reinforced hydrogel.

In some examples, the outer surface of the hydrogel may be formed to besmooth (e.g., to have a roughness of less than 30 microns). For example,the methods described herein may include mechanically polishing an outersurface of the hydrogel to a roughness of less than 30 microns. In somecases, the outer surface may be formed smooth by molding, includingmolding the heated polymer using a smooth mold. For example,infiltrating the nanofiber network with hydrogel may include molding thehydrogel so that an outer surface of the hydrogel has a roughness ofless than 30 microns. Molding the outer surface may also allow amanufacturer to form the outer surface into any desired shape. Forexample, the shape may be concave, convex, saddle shaped, etc. Anydesired shape (and smoothness) may be formed, e.g., by molding and/orpolishing.

In any of these methods securing the (e.g., dry) nanofiber network mayinclude securing, such as clamping and/or cementing, a freeze-driednanofiber network. As mentioned above, any of these devices and methodsmay use a dry nanofiber network that comprises a cellulose nanofibernetwork. The dry nanofiber network may comprise at least one of:electrospun polymer nanofibers, poly(vinyl alcohol) (PVA) nanofibers,aramid nanofibers, Aramid-PVA nanofibers, wet-spun silk proteinnanofiber, chemically crosslinked cellulose nanofiber, orpolycaprolactone (PCL) fibers.

Any of the methods described herein may include rehydrating thenanofiber network. Including rehydrating it after it has been secured tothe implant surface.

Any of these methods may include mineralizing at least a portion of thenanofiber network adjacent to the surface.

Described herein are implants for knee resurfacing or partial kneeresurfacing. For example, a top bearing surface of the implant mayinclude a hydrogel having a water content of at least 20 wt %, in whichthe hydrogel is attached to a metallic base with a shear strengthexceeding 0.2 MPa.

Any of the methods described herein may include mechanically polishingan outer surface of the hydrogel (e.g., cellulose-reinforced hydrogel)to a roughness of less than 50 microns (e.g., less than 50 microns, lessthan 40 microns, less than 30 microns, less than 25 microns, less than20 microns, less than 15 microns, less than 10 microns, etc.).Mechanically polishing may include abrading the hydrogel that isattached to the surface as described herein with a fine grit sandpaperor equivalent.

Any of these methods may include rehydrating the nanofiber network. Thenanofiber network may be rehydrated before impregnating with thehydrogel or the impregnation may rehydrate the nanofiber network.

For example, described herein are implants, comprising: an implant bodyhaving a top bearing surface; an anchoring base (which may extend from aback of the top bearing surface); and a cellulose-reinforced hydrogelcomprising: a cross-linked cellulose nanofiber network secured over thetop bearing surface of the implant body; and an interstitial hydrogelportion within interstitial regions of the cross-linked cellulosenanofiber network, wherein the interstitial hydrogel portion has acrystallinity of 20% or greater. The interstitial hydrogel portion maypolyvinyl alcohol (PVA). The cellulose-reinforced hydrogel may compriseat least 20% by weight of water. The cellulose-reinforced hydrogel mayhave a tensile strength exceeding 40 MPa. The cross-linked cellulosenanofiber network may be chemically cross-linked. The cross-linkedcellulose nanofiber network may comprise bacterial cellulose (BC). Thecellulose-reinforced hydrogel may have a compressive strength exceeding59 MPa. The cross-linked cellulose nanofiber network may be secured overthe top bearing surface by a clamp. In some examples the cross-linkedcellulose nanofiber network comprises one or more sheets of bacterialcellulous (BC) held over the top bearing surface by a clamp secured to alip or rim of the top bearing surface. The clamp may be used to securethe cross-linked cellulose nanofiber network without the need for epoxy.Alternatively any of these implants may include an adhesive.

Also described herein are methods of forming an implant having acellulose-reinforced hydrogel, comprising: attaching a cross-linkedcellulose nanofiber network to a top bearing surface of the implant;infiltrating a hydrogel component within interstitial regions of thecross-linked cellulose nanofiber network to form thecellulose-reinforced hydrogel; and annealing the cellulose-reinforcedhydrogel so that a crystalline content of the hydrogel component has acrystallinity of 20% or greater. The hydrogel component may comprisepolyvinyl alcohol (PVA). Annealing the cellulose-reinforced hydrogel mayinclude heating the cellulose-reinforced hydrogel. For example,annealing the cellulose-reinforced hydrogel may comprise heating thecellulose-reinforced hydrogel to decrease a water content of thecellulose-reinforced hydrogel. In some examples the cellulose-reinforcedhydrogel is heated to a temperature ranging from 90-140° C. Annealingthe cellulose-reinforced hydrogel may comprise rehydrating thecellulose-reinforced hydrogel. Rehydrating the cellulose-reinforcedhydrogel may comprise increasing a water content of thecellulose-reinforced hydrogel to at least 20 wt %. Any of these methodsmay include removing excess of the hydrogel component from a surface ofthe cross-linked cellulose nanofiber network. For example, excess of thehydrogel component may be removed by hand or by molding thecellulose-reinforced hydrogel. In any of these examples the cross-linkedcellulose nanofiber network may comprise bacterial cellulose (BC). Insome examples attaching the cross-linked cellulose nanofiber network tothe top bearing surface comprises clamping the cross-linked cellulosenanofiber around a periphery of the top bearing surface.

Also described herein are implants for a knee resurfacing, the implantcomprising: a top bearing surface comprising a cellulose-reinforcedhydrogel comprising: a cellulose nanofiber network; and a hydrogelcomponent impregnated in the cellulose nanofiber network, wherein thehydrogel component has a crystallinity of 20% or greater. The hydrogelcomponent may comprise polyvinyl alcohol (PVA). The cellulose-reinforcedhydrogel may comprise at least 20% by weight of water. Thecellulose-reinforced hydrogel may have a tensile strength exceeding 40MPa. The cellulose-reinforced hydrogel may be attached to a metallicbase of the top bearing surface with a shear strength exceeding 0.2 MPa.The top bearing surface may have a coefficient of friction (COF) that isnot statistically greater than that of cartilage.

In general, the methods and apparatuses described herein may be usedwith any of the methods, apparatuses and compositions described inInternational Patent Application No. PCT/US2021/040031, titled“NANOFIBER REINFORCEMENT OF ATTACHED HYDROGELS,” filed on Jul. 1, 2021,which is herein incorporated by reference in its entirety.

All of the methods and apparatuses described herein, in any combination,are herein contemplated and can be used to achieve the benefits asdescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the features and advantages of the methods andapparatuses described herein will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,and the accompanying drawings.

FIG. 1A is an illustration of an exemplary process for attachment of ahydrogel to a porous base by a Nanofiber-Enhanced STicking (NEST)method. In this example, a nanofibrous sheet (e.g., bacterial cellulose)is attached to a surface (e.g., a porous base such as porous titanium)with an adhesive (e.g. α-TCP cement), after which the hydrogelcomponents are infiltrated into the nanofibrous sheet.

FIG. 1B shows an example of a hydrogel bonded to a titanium plug.

FIG. 1C shows an SEM image of a surface of an exemplary freeze-driedbacterial cellulose sheet.

FIGS. 2A-2D show various hydrogel samples before and after annealing andrehydration to illustrate the effects on the morphology of differenthydrogel compositions.

FIGS. 3A-3E are graphs illustrating effects of annealing on themechanical properties of various hydrogel compositions: FIG. 3Aillustrates a tensile stress-strain curve for PVA hydrogels annealed atdifferent temperatures; FIG. 3B illustrates a compressive stress-straincurve for PVA hydrogels annealed at different temperatures; FIG. 3Cillustrates tensile strength and moduli of PVA annealed at differenttemperatures; FIG. 3D illustrates compressive stress at 0.8 strain andmoduli of PVA annealed at different temperatures; and FIG. 3Eillustrates crystallinity and solid content weight fraction of PVAannealed at different temperatures.

FIGS. 4A-4E are graphs illustrating effects of annealing on themechanical properties of BC-PVA hydrogel. FIG. 4A illustrates a tensilestress-strain curve for BC-PVA hydrogels annealed at differenttemperatures; FIG. 4B illustrates a compressive stress-strain curve forBC-PVA hydrogels annealed at different temperatures; FIG. 4C illustratestensile strength and moduli of BC-PVA annealed at differenttemperatures; FIG. 4D illustrates compressive stress at 0.8 strain andmoduli of BC-PVA annealed at different temperatures; and FIG. 4Eillustrates crystallinity and solid content weight fraction of BC-PVAannealed at different temperatures.

FIGS. 5A and 5B are graphs illustrating effects of PAMPS on an annealedBC-PVA hydrogel: FIG. 5A illustrates tensile strength, tensile moduliand solid content weight fraction of BC-PVA-PAMPS hydrogels that weremade with solutions containing different concentrations of the AMPSmonomer, where BC-PVA samples were annealed before infiltration of AMPS;and FIG. 5B illustrates compressive strength and moduli of BC-PVA-PAMPShydrogels.

FIGS. 6A-6D illustrate various aspects of measuring wear and coefficientof friction (COF) of various hydrogel compositions and comparing suchmeasurements against cartilage: FIG. 6A illustrates a schematic for howthe wear and COF of hydrogels versus cartilage was measured; FIG. 6Billustrates Micro-CT cross-section images of the cartilage and hydrogelsamples; FIG. 6C illustrates the wear depth of cartilage and hydrogelsamples after 106 cycles under 1 MPa of pressure, a spin rate of 100mm/s, and with FBS as the lubricant; and FIG. 6D illustrates the COFbetween cartilage and the hydrogels during the tests over 24 hours.

FIGS. 7A-7C illustrate various aspects of measuring wear of varioushydrogel compositions and comparing such measurements against cartilage:FIG. 7A illustrates a schematic for how the wear of cartilage versushydrogels was measured; FIG. 7B illustrate Micro-CT cross-sectionimages; and FIG. 7C illustrates the wear depth of cartilage and hydrogelsamples after 106 cycles under 1 MPa of pressure, a spin rate of 100mm/s, and with FBS as the lubricant.

FIGS. 8A-8D illustrate various aspects of shear strength testing ofvarious hydrogel composition and cartilage: FIG. 8A illustrates resultsfor shear testing of pig cartilage and hydrogels secured to metal pinswith adhesive and shape memory alloy clamps; FIG. 8B illustrates animage of an osteochondral plug extracted from a pig knee after testingto failure; FIG. 8C illustrates an image of a BC-PVA-PAMPS hydrogel(fabricated with a freeze-thaw process) after testing to failure; andFIG. 8D illustrates an image of a BC-PVA hydrogel (fabricated byannealing at 90° C. and rehydrated) after testing to failure.

FIGS. 9A-9F show front and side views of an annealed BC-PVA hydrogelincorporated in an implant for partial knee resurfacing before and afterundergoing mechanical stress.

FIGS. 10A and 10B schematically illustrate examples of implantsincluding a hydrogel attached (e.g., forming the surface) as describedherein.

FIG. 11 illustrates an exemplary method of forming and attaching ahydrogel surface.

FIG. 12A is an image illustrating one example of a method of attaching ahydrogel to a metallic plug, including using a clamp (e.g., a shapememory alloy clamp).

FIGS. 12B and 12C illustrate examples of a fixture that may be used foraligning forming the materials described herein (e.g., aligning the BC,including a rod, cut BC, and ring clamp) as described herein: FIG. 12Bshows a perspective view of the fixture; and FIG. 12C shows a sectionalview through the fixture.

FIG. 12D is an image showing an exemplary sheet of bacterial cellulose(BC) cut (e.g., with legs or crenellations) for wrapping over the edgeof a bearing surface (e.g., metal rod, head, etc.).

FIG. 12E is diagram denoting the diameter D, length L, and width Wdimensions of one example of a sheet.

FIG. 13 is an example of one test fixture that may be used to testsearing of cartilage off of bone and/or a hydrogel material off of atest rod.

FIG. 14A shows an example of a process for attaching the BC-PVA-PAMPShydrogel to a titanium implant for treatment of osteochondral defects.FIGS. 14B and 14C show repair of an implant.

FIGS. 15A-15D show hydrogel samples before and after annealing andrehydration. All samples were annealed at 90° C. for 25 hours andrehydrated in PBS solution for 24 hours at 23° C. (A) A sample of BCwithout PVA. (B) A sample of BC that was annealed in a solution of 10wt. % PVA. (C) A sample of 40 wt. % PVA. (D) A BC sample that wasinfiltrated with 40 wt. % PVA in a hydrothermal bomb for 24 hours at120° C. before annealing and rehydration.

FIG. 16A shows an FTIR spectra of BC-PVA, annealed BC-PVA and annealedPVA hydrogels. FIG. 16B shows a zoomed-in region highlighting the shiftof the hydroxyl peak. FIG. 16A shows an increase in hydrogen bondingthat occurs upon annealing.

FIGS. 17A-17C show DSC thermograms for (A) freeze-thawed and annealedPVA hydrogels, (B) freeze-thawed and annealed BC-PVA hydrogels, and (C)annealed BC-PVA and annealed BC-PVA-PAMPS. The concentration of the AMPSsolution used to make the BC-PVA-PAMPS was 10 wt. %.

FIG. 18 show a DSC thermogram for an annealed BC-PVA hydrogel sampledemonstrating how the peak was integrated.

DETAILED DESCRIPTION

Described herein are hydrogel compositions for the long-term repair ofcartilage. The hydrogels have a crystalline structure that imparttensile and compressive strengths to the hydrogels that equal or exceedthat of cartilage. The hydrogels may be incorporated in a nanofibernetwork (e.g., cellulose) to increase wear properties and/or tofacilitate attachment to an implant body. The hydrogels are found towithstand the high compressive and shear stresses associated movement ofthe knee joint, and are thus well-suited for implementation on kneeimplants. The hydrogels may be characterized by one or more attributesand properties, such as crystalline structure, tensile strength,compressive strength, water content, coefficient of friction (COF),and/or other attributes and properties.

The methods of forming hydrogel implants described herein can be used tocreate hydrogel-coated orthopedic implants with surfaces that mimic orimprove on the mechanical and/or tribological properties of cartilage.Previously methods of forming implants with hydrogels comprising abacterial cellulose (BC) network infused with polyvinyl alcohol (PVA)and poly(2-acrylamido-2-methyl-1-propanesulfonic acid sodium salt(PAMPS), referred to as BC-PVA-PAMPS hydrogels, are described inInternational Patent Application No. PCT/US2021/040031, which isincorporated herein by reference in its entirety. In preparing theBC-PVA-PAMPS hydrogel, a freeze-thaw method is used to gel a PVA-watermixture after infiltration into the BC. This freeze-thaw gelation stepwas used to increase the strength of the PVA hydrogel, and to preventdissolution of the PVA in a following PAMPS infiltration step. Theincrease in strength upon freezing and thawing the PVA is attributed tocrystallization of the PVA chains and phase segregation.

Methods described herein improve upon these previous methods byenhancing the mechanical properties of the reinforced hydrogels, therebyimproving performance of the implant, even when subjected to high impactand shear forces. For example, the tensile and compressive strength ofthe reinforced hydrogel may be increased up to and beyond that ofcartilage. The methods involve a mechanical strengthening process thatincreases the crystallinity and decreases the water content of PVArelative to the freeze-thaw process. The mechanical strengtheningprocess may include drying, annealing, and/or rehydrating of thehydrogel. When implemented on a hydrogel, the crystalline structurewithin the hydrogel may be increased despite being interdigitated withinthe fibrous network. In addition, the hydrogel may be substantiallybubble-free and crack-free after undergoing the crystal restructuringprocess.

FIGS. 1A-1C illustrate an example of an apparatus in which a hydrogelhas been bonded to an implant surface as described herein. The hydrogelmay be coupled to the implant surface by first attaching a layer ofnanofibrous material, such as cellulous (e.g., bacterial cellulous) toan implant base using an adhesive (e.g., cement). The nanofibrousmaterial may be dry (e.g., before attaching to the implant base). Theattaching surface of the implant base may be porous, for example, toenhance adhesion. The nanofibrous layer may then be infiltrated with ahydrogel component. In this way, the nanofibrous portion may be securedwith an adhesive (e.g., cement) that can penetrate and secure the porousbacterial cellulose network to the surface and may create aninterdigitating bond without the interference of water. Once thehydrogel component is infiltrated within the nanofibrous network, thereinforced hydrogel may be processed (e.g., annealed) to change thecrystalline structure of the hydrogel component, thereby enhancingmechanical properties of the reinforced hydrogel.

For example, in FIG. 1A, the nanofibrous portion is bacterial cellulose(BC) 101 that is applied to the prepared surface of the implant (shownin this example as a titanium base, having pores) 103. A cement (e.g.,any appropriate medical or dental grade cement may be used) is applied,and secures the dry bacterial cellulose to the implant surface.Thereafter the hydrogel component may be infiltrated into thenanofibrous portion, resulting in the complete hydrogel 107 attached tothe base 103 via the bacterial cellulose 101. The reinforced hydrogel107 then undergoes a crystal restructuring process to enhance itsmechanical properties.

FIG. 1B shows an example of a titanium implant (e.g., plug) to which acellulous-reinforced hydrogel has been attached, as described herein. Inthis example, the nanofibrous portion (e.g., BC) of the hydrogel isbonded via an adhesive to the porous surface of the implant, and thehydrogel is linked to the nanofibrous portion. Any appropriate adhesive(e.g., cement) may be used to adhere the nanofibrous portion of thehydrogel to the surface of the implant. In some variation the cement isα-tricalcium phosphate (α-TCP), a hydroxyapatite-forming cement that maybe used for attachment of the hydrogel due to its biocompatibility,osteoconductivity, and shear strength, which may exceed that ofcyanoacrylate. In some cases, α-TCP is combined with phosphoserine (PPS)to promote adhesion. In some cases, the hydroxyapatite is reinforcedwith stainless-steel powder (SSP) (e.g., with an average particle sizeof 150 μm) to hinder crack propagation. As will be described in greaterdetail below, in some examples an adhesive is not used, and thenanofiberous portion is secured to the bearing surface by a mechanicalmeans (such as a clamp).

As described herein, the nanofibrous portion (e.g., BC) may be treatedto dried (e.g., freeze-dried) to increase adhesion to the nanofibers.FIG. 1C is a scanning electron microscope (SEM) image of the surface ofan exemplary freeze-dried piece of BC, which shows that it consists ofmany nanoscale fibers that present a large surface area for attachmentwith an adhesive. In some examples, multiple freeze-thaw cycles areperformed, which may increase tensile strength (once the hydrogel isinfused therein) and/or increase the shear strength of the adhesion ofthe reinforced hydrogel to the implant base.

As mentioned above, a previous approach to creating acartilage-equivalent hydrogel involves infiltrating a bacterialcellulose (BC) nanofiber network with polyvinyl alcohol (PVA) andpoly(2-acrylamido-2-methyl-1-propanesulfonic acid sodium salt) (PAMPS).This hydrogel exhibited a tensile strength of 22.6 MPa and a compressionstrength of 20 MPa. In comparison, the range of tensile and compressionstrengths reported for human cartilage are 8.1-40 MPa and 14-59 MPa,respectively. Thus, there is room to improve the strength of hydrogelsto be at the higher end of the range of strengths reported forcartilage, or to exceed cartilage in strength, while having a similarmodulus, coefficient of friction, and resistance to wear as cartilage.Given the higher tensile strength of annealed PVA relative tofreeze-thawed PVA, tests were performed to determine whether changingfrom a freeze-thaw to annealing process can improve the mechanicalstrength of a BC-PVA-PAMPS hydrogel while retaining adequate controlover the hydrogel shape and defect content. Given the tensile strengthof a BC-PVA-PAMPS hydrogel (22.6 MPa), is already similar to the tensilestrength of a PVA hydrogel made by annealing (20 MPa), it was notobvious that switching to the annealing process for a BC-reinforcedhydrogel would yield further improvements in the mechanical strength. Inaddition, the presence of BC or PAMPS could potentially interfere withthe crystallization of PVA that occurs during the annealing process,thereby hindering the improvement in mechanical strength that occurs asa result of crystallization. It was also not clear whether it would bepossible to obtain high-quality, bubble-free, crack-free samples afterannealing PVA reinforced with BC. Obtaining samples that are as free ofdefects as possible may be necessary to maximizing the mechanicalstrength of the hydrogel. Finally, it was unclear whether the lowerwater content of the annealed hydrogel might cause the COF and opposingsurface wear to be too high.

As demonstrated herein, reinforcement of annealed PVA with BC leads to a3.2-fold improvement in the tensile strength (from 15.6 to 50.5 MPa) anda 1.7-fold increase in the compressive strength (from 56.7 to 95.4 MPa).The highly crystallized BC-PVA hydrogel that results from annealing isthe first hydrogel with a tensile and compressive strength that exceedsthat of cartilage. Reinforcement of the PVA with BC may essentiallyeliminate the deformation and bubbles that would otherwise occur duringannealing. When tested against cartilage, annealed BC-PVA wore anopposing cartilage surface to the same extent as cartilage and was threetimes more resistant to wear than cartilage. The COF of BC-PVA againstcartilage was equivalent to that of cartilage against cartilage. Incontrast to results with freeze-thawed BC-PVA, addition of PAMPS to theannealed BC-PVA decreased the tensile strength of the hydrogel due to aloss of crystallized PVA and an increase in water content. The improvedtensile strength of annealed BC-PVA enabled it to attach to a metal basewith a shear strength 68% greater than the shear strength of cartilageon bone. The high strength, high wear resistance, and low COF ofannealed BC-PVA make it an excellent material for replacing damagedcartilage.

The tests and measurements performed and described herein on varioushydrogel compositions demonstrate how certain hydrogel compositions thatundergo one or more strengthening processes, such as an annealingprocess, may increase the strength of a hydrogel to the upper limits orexceeding that of cartilage while attaining other characteristics (e.g.,coefficient of friction) similar to that of cartilage.

One potential disadvantage of annealing a hydrogel is that the hydrogelmay develop bubbles and cracks, especially as the sample thicknessincreases or water content increases. FIGS. 2A-2D show various hydrogelsamples before and after annealing and rehydration to illustrate theeffects on the morphology of different hydrogel compositions. Inparticular, these samples illustrate the effect of the hydrogelcomposition on the shape of the sample after drying, annealing andrehydration. All samples were annealed at 90° C. for 25 hours andrehydrated in phosphate-buffered saline (PBS) solution for 24 hours at23° C. FIG. 2A shows a sample of BC (without PVA), demonstrating how theBC sample became wrinkled and folded at the edges after annealing andrehydration. FIG. 2B shows a sample of BC that was annealed in asolution of 10 wt % PVA. As shown, the BC annealed in 10 wt % PVAsolution also demonstrating substantial deformation. A PVA layer formedon top of the BC after annealing contains a large number of bubbles andeasily delaminates from the BC film. The bubbles may be the result ofthe evaporation of water. FIG. 2D shows a sample 40 wt % PVA before andafter annealing, demonstrating that such hydrogel also formed a largenumber of bubbles and deformed during the annealing process.

FIG. 2E shows a BC sample that was infiltrated with 40 wt % PVA in ahydrothermal bomb for 24 hours at 120° C. before annealing andrehydration. As shown, reinforcement of 40 wt % PVA with BC allowed thehydrogel to retain its shape with little to no deformation afterannealing. This lack of deformation may be attributed to the highersolid content and tensile modulus of the BC-reinforced PVA. That is, thenano scale network of the BC layer appears to suppress the formation ofthe large bubbles that are visible in the 40 wt % PVA sample (FIG. 2C).A comparison of FIG. 2B to FIG. 2D indicates that the approach ofinfiltrating a high concentration of PVA into BC in a hydrothermal bomb,followed by removal of excess PVA from the BC surface, results in a moreuniform hydrogel than if a BC sample is placed in a more dilute PVAsolution that is concentrated via drying. These results demonstratethat, unlike BC alone, PVA alone, or the combination of BC with a 10 wt% of PVA, the BC infiltrated with 40 wt % PVA could retain its shape andremain relatively free of bubbles and other defects after annealing.

FIGS. 3A-3E illustrate the effects of annealing on the mechanicalproperties of various hydrogel compositions, where the effects ofannealing on a PVA hydrogel were analyzed as a reference point. The PVAwas fully hydrolyzed with a molecular weight of 145,000 g mol-1. A 40wt. % PVA solution was dried at 90° C. for 24 hours, annealed at 90° C.,120° C. or 140° C., and then placed in a 0.15 M PBS solution for 24hours for rehydration. PVA samples that underwent a freeze-thaw cyclewere tested for comparison. FIGS. 3A and 3B show that annealing thehydrogel dramatically increased the tensile and compressive strengthrelative to samples that had undergone a freeze-thaw cycle. FIGS. 3C and3D show that, relative to the freeze-thaw process, annealing increasedthe tensile strength by 60 times (from 0.26 to 15.6 MPa) and thecompressive strength by 9 times (from 14.8 to 140.8 MPa). Increasing theannealing temperature from 90° C. to 140° C. led to an increase in thetensile strength and modulus. The increase in strength and modulus hasbeen ascribed to the increase in the crystallinity and solid content ofthe hydrogel after annealing. FIG. 2E confirms that the crystallinityand solid content of the annealed PVA hydrogels are much greater thanthat of a freeze-thawed PVA hydrogel. For example, a PVA hydrogel madevia the freeze-thaw process has an overall solid content of 0.09 and aPVA crystallinity of 0.21, whereas a PVA hydrogel made via annealing at90° C. has an overall solid content of 0.42 and a PVA crystallinity of0.58. The crystallites formed during annealing strengthens the otherwiseamorphous PVA by acting as tough cross-links that redistribute appliedstresses and hinder crack propagation. The crystallites also increasethe solid content and strength of the hydrogel by reducing the amount ofwater taken up by the PVA when it is soaked in 0.15 M PBS afterannealing.

FIGS. 4A-4E illustrate the effect of annealing on a BC-PVA hydrogel. Thesame annealing process to PVA hydrogel described above with reference toFIGS. 3A-3E was applied to BC-PVA hydrogels. As with the PVA hydrogels,the BC-PVA hydrogels were dried at 90° C. for 24 hours, annealed at 90°C., 120° C. or 140° C., and then placed in a 0.15 M PBS solution for 24hours for rehydration. FIGS. 4A and 4C show the tensile strength of theannealed BC-PVA hydrogels reached 50.4 MPa, an increase of 4.6 timesrelative to the BC-PVA that went through a freeze-thaw cycle, and anincrease of 3.2 times relative to annealed PVA that was not reinforcedwith BC. FIGS. 4B and 4D show the compressive strength increased from55.32 MPa to 95.35 MPa after annealing. Similar to the PVA hydrogel,this dramatic increase in strength can be attributed to the increase incrystallinity and solid content after annealing. FIG. 3E shows thecrystallinity of the BC-PVA hydrogel increased from 0.07 after afreeze-thaw cycle to 0.4 after annealing. The solid weight fraction ofthe BC-PVA hydrogel increased from 0.11 after a freeze-thaw cycle to0.53 after annealing. These results shows that PVA can still formcrystallites within the nanofibrous BC network, and that thesecrystallites increase the solid content and strength of the hydrogel.

FIGS. 5A and 5B illustrate the effect of PAMPS on an annealed BC-PVAhydrogel. As mentioned above, it was previously found that theincorporation of PAMPS into a BC-PVA hydrogel made with a freeze-thawcycle resulted in an increase in the tensile and compressive strength ofthe hydrogel. Thus, PAMPS was incorporated into an annealed BC-PVAhydrogel to determine the effect of the addition of PAMPS. As shown inFIG. 5A, the addition of PAMPS into the annealed BC-PVA hydrogel led toa decrease in the solid content relative to BC-PVA alone, from 0.53 to0.37. Differential scanning calorimetry (DSC) thermograms show thatafter the addition of 10 wt % PAMPS, the peak from melting crystallinePVA disappeared, indicating the addition of PAMPS destroys the PVAcrystallites that form during the annealing process. The decrease insolid content and loss of crystallinity upon addition of PAMPS led to adecrease in the tensile strength (from 48.9 MPa to 20.8 MPa), tensilemodulus (from 444.8 MPa to 150.5 MPa) and compressive strength (from98.1 MPa to 56.0 MPa in FIG. 5B) of the hydrogel. The increase in watercontent of the hydrogel and loss of strength was likely due to the factthat PAMPS is a negatively charged polymer, and this negative chargeresults in an osmotic pressure that swells the hydrogel with water.

FIGS. 6A-6D illustrate various aspects of measuring wear and coefficientof friction (COF) of various hydrogel compositions and comparing suchmeasurements against cartilage. FIG. 6A is a schematic illustration ofan example of how a wear test is performed. A pin-on-disc configurationdescribed in Example 11 below for testing the wear of hydrogels in fetalbovine serum (FBS) was used. A porcine cartilage plug was rotatedagainst the hydrogel surface 106 times under 1 MPa of pressure and at aspeed of 319 rotations per minute (maximum linear velocity was 100 mms⁻¹). The wear resistance of a potential replacement for cartilageexceeds that of cartilage to ensure durability and minimize thegeneration of wear debris that could potentially cause an adversebiological reaction. The wear resistance of a BC-PVA-PAMPS hydrogel waspreviously shown to be equivalent to that of cartilage and to besuperior to PVA or PVA-PAMPS when tested against a stainless-steel pin.These hydrogels were made by applying a freeze-thaw cycle to crystallizethe PVA. FIGS. 6B-6D compare the wear resistance of PVA-based hydrogels(PVA, BC-PVA, and BC-PVA-PAMPS) that have been dried and annealed at 90°C. to that of porcine cartilage when tested against a porcine cartilageplug in FBS.

FIG. 6B shows cross-sectional Micro-CT images of the hydrogels that wereacquired in the center of the wear mark to measure the maximum weardepth. FIG. 6C compares the wear depth of the hydrogels and cartilage.The wear depth of the BC-PVA hydrogel with 0% AMPS was 70.1 μm. Theaddition of 20% AMPS decreased the mean wear depth to 65.9 μm, but thedifference between the 0% and 20% AMPS samples was not statisticallysignificant. This comparison illustrates that the negative charge andhigher water content caused by incorporating PAMPS into an annealedBC-PVA hydrogel does not significantly improve the wear resistance. Bothof these values were 3 times lower than the wear depth on the cartilagesample, which was 227.8 μm. The wear depth for annealed and rehydratedPVA was 301.0 μm, four times greater than either BC-PVA sample. Theseresults indicate the presence of BC in the hydrogel can dramaticalimprove the wear resistance of an annealed PVA hydrogel to be superiorto that of cartilage.

The COF was recorded during the wear test, as shown in FIG. 6D.Cartilage maintained a constant COF of 0.020 during the test. The COF ofBC-PVA decreased during the test from 0.040 to 0.021. The BC-PVAhydrogel with 20% AMPS had a similar COF as that without AMPS. Incontrast, the COF of PVA increased dramatically during the test, from0.033 to 0.135. Previous work has similarly demonstrated the COF of PVAagainst cartilage increases over time while the COF of cartilage againstcartilage is constant. The increase in the COF for a PVA-Cartilageinterface has been ascribed to transfer of damaged PVA to the cartilagesurface, which, in turn, decreases the ability of the cartilage surfaceto maintain a lubricating water layer. The incorporation of BC into PVAclearly inhibits damage of the hydrogel, allowing it to maintain a lowcoefficient of friction similar to that cartilage during the wear test.The presence of AMPS in the hydrogel does not appear to be necessary formaintaining a low COF and high resistance to wear.

The materials used for cartilage replacement on one side of the joint,i.e., on the femoral condyle, should not cause wear of cartilage on theopposing surface, i.e., the tibial plateau. Traditional orthopedicmaterials like cobalt-chrome and ultra-high molecular-weightpolyethylene are known to damage an opposing cartilage surface to agreater extent than hydrogels due to the higher COF and hardness oftraditional orthopedic materials. To assess the wear caused by BC-PVAand BC-PVA-PAMPS hydrogels on cartilage, hydrogel plugs were created forwear testing (see Example 7 below). The hydrogel plugs were pressedagainst cartilage samples (see FIG. 7A) with 1 MPa of pressure, androtated 106 times at a speed of 319 rotations per minute (the maximumlinear velocity at the circumference of the pin was 100 mm s⁻¹).

FIG. 7B shows cross-sectional Micro-CT images of the cartilage samplesthat were acquired in the center of the wear mark to measure the maximumwear depth. FIG. 7C compares the wear depth on cartilage caused by thehydrogels or cartilage. The wear caused by the BC-PVA on cartilage(247±16 μm) was not significantly different from the wear caused bycartilage on cartilage (228±12 μm). The addition of PAMPS into theBC-PVA reduced the wear on the opposing cartilage surface to 81±27 μm,significantly below the wear of cartilage on cartilage.

FIGS. 8A-8D show various aspects of shear strength testing of varioushydrogel composition and cartilage. It was hypothesized that increasingthe tensile strength of the hydrogel should also increase the shearstrength. In order to be used for a cartilage replacement material, asynthetic hydrogel should be secured into a defect site with the sameshear strength as the junction between cartilage and bone. One way toaccomplish this goal is to have hydrogels that directly attach to boneor cartilage with sufficient strength. Alternatively, the hydrogel canbe attached to a metallic base, such as titanium, which has the abilityto integrate with bone. As mentioned above, the ability to attachBC-PVA-PAMPS hydrogels to a metallic base with a shear strengthequivalent to the cartilage-bone interface, as described inPCT/US2021/040031. Previous tests indicated the strength of hydrogelattachment was limited by the tensile force required to fracture thehydrogel that is curved over the edge of the metallic base. Thus,increasing the tensile strength of the hydrogel should in turn increasethe shear strength with which the hydrogel is attached to a metallicbase.

The setup used for shear testing is described in Example 7 and 12 below.FIGS. 8A-8D show the results for shear testing a plug of porcinecartilage on bone extracted from a pig knee, testing of a BC-PVA-PAMPShydrogel made with the previous freeze-thaw process, and testing aBC-PVA hydrogel annealed at 90° C. and then rehydrated. Both of thehydrogels are attached to stainless-steel rods with a combination ofRelyX Ultimate cement and a shape memory alloy ring. The BC-PVA shearstrength of 1.98 is significantly greater than that of porcine cartilage(p-value from one-way ANOVA is <0.05). The average value of the shearstrength for BC-PVA is also 40% greater than that of BC-PVA-PAMPS, butthe error in the measurements is such that the difference in thesevalues is not statistically significant. Comparison of the sample afterfailure show that while pig cartilage was sheared completely off of theunderlying bone, both BC-PVA-PAMPS (made with the freeze-thaw process)and BC-PVA (made by annealing at 90° C., followed by rehydration) werefractured on one side of the cylindrical sample but remained attached.These results show that the shear strength of attachment for theannealed BC-PVA is greater than that of pig cartilage.

Described above are tensile strength, compression strength and shearstrength of BC-reinforced hydrogels attached to a metal pin with adiameter of 5.2 mm. While this size is convenient for testing, such adiameter is too small to serve as an implant for partial kneeresurfacing. In addition, the samples lacked the curvature necessary tomimic the natural curvature of the femoral condyle. FIGS. 9A-9F showapplication of an annealed BC-PVA hydrogel on an implant for partialknee resurfacing to demonstrate the ability of the hydrogel to attach toa metal base with a size and shape representative of an implant forpartial knee resurfacing.

FIGS. 9A and 9B show the implant prior to mechanical testing. Theimplant sample is 20 mm in diameter with a radius of curvature of 20 mm.An implant diameter of 20 mm is a typical size used for an osteochondralallograft, and a 20 mm radius of curvature is within the range oftypical curvatures for the femoral condyle. The peak force on the kneeduring jogging has been measured to be 5551 N for a body weight of 100kg. The tibiofemoral contact area has been measured to be 1500 mm 2 at3100 N. The peak stress on the contacted area of the knee during joggingis 3.7 MPa based on the assumption that the contact area will notincrease at a higher force. FIGS. 9C and 9D show the implant after beingsubjected to a compression stress of 16 MPa, 4.3 times greater than thepeak physiological force on the femoral condyle. After this test, therewere no signs of fracture or damage on the surface of the hydrogel. Thistest indicates an implant created with the annealed BC-PVA hydrogel canwithstand the compressive forces in the knee without fracture. Peakanterior shear forces in the knee for walking have been measured to be30% of body weight, or 294 N for a 100 kg individual. This is thehighest shear force measured in the knee for any investigated dailyactivity. The tibiofemoral contact area has been measured to be 1500 mm2 at 3100 N, which is approximately equivalent to the peak normal forceduring walking for an individual with a body weight of 100 kg. As thepeak normal force coincides with the peak shear force, we can use thistibiofemoral contact area to calculate that the peak shear stressexperienced by cartilage during walking is 0.2 MPa (294 N÷1 500 mm²).Shear testing on the implant indicated failure did not occur until astress of 0.9 MPa was applied. Since the implant can withstand shearloads 4.5 times greater than that experienced by cartilage in the knee,this result shows the annealed BC-PVA hydrogel and method of attachmenthave sufficient strength for creation of an implant for partial kneeresurfacing.

An implant for partial knee resurfacing may be relatively large and maybe curved to mimic the natural curvature of the femoral condyle. FIGS.14A-14C shows images of an implant 20 mm in diameter with a radius ofcurvature of 20 mm. An implant diameter of 20 mm is a typical size usedfor an osteochondral allograft, and a 20 mm radius of curvature iswithin the range of typical curvatures for the femoral condyle. In thisexample, five pieces of BC were cut into shapes with 8 octagonal legs toenable the BC to fold over the edge of the implant. A 0.25-mm-thickcoating of commercially pure titanium was applied to the stem of theimplant and underneath the base with a plasma spray process in order toimprove integration with bone. FIGS. 14B and 14C show an example of howsuch an implant would be used to resurface the knee. FIG. 14B shows anexample of a cartilage defect. The surgeon would drill out a hole overthe defect site that is complementary to the shape of thehydrogel-capped implant. The hydrogel-capped implant would then bepressed into the hole to replace the damaged cartilage.

The methods and apparatuses described herein may be used to reinforce anannealed PVA hydrogel with BC to provide, for the first time, a hydrogelwith a compression and tensile strength greater than cartilage.Annealing increased the tensile strength of BC-PVA by 5 times and thecompressive strength by 1.8 times relative to a freeze-thaw process dueto the greater crystallization and lower water content that was achievedby annealing. Reinforcement of PVA with BC lowered the wear of thehydrogel by 4 times relative to PVA alone, and 3 times relative tocartilage. The annealed BC-PVA hydrogel caused a minimal amount ofopposing surface wear, similar to what was caused by cartilage onitself. Attachment of the BC to a metal plug via an adhesive and/orclamp, followed by infiltration and annealing of the PVA, enabledattachment of the BC-PVA hydrogel to a metal backing with a shearstrength greater than the attachment of cartilage to bone. Theseadvances in hydrogel strength and attachment enable the creation of animplant with a hydrogel surface and titanium backing that can enabledurable resurfacing of damaged cartilage in an articulating joint.

As used herein, an implant may have any appropriate structure forimplanting into a body. In some (non-limiting) examples, the implantsmay have a shape that allows them to be implanted into bone, with ahydrogel attached to an outward-facing side. For example, FIGS. 10A and10B illustrate examples of implants to which a hydrogel has beenattached, as described herein. In FIG. 10A, the implant includes a base1001 (e.g., a titanium base) having an elongate pin-shape that may be,for example, 2 mm×7 mm (tapering to about 1.5 mm at about 3 mm from theend). The base may include one or more channels, openings, passages,etc. for ingrowth of bone. The implant also includes a top portion 1005that may be curved (e.g., with a single curvature or a double-curvature.For example, the surface may be curved with a radius of curvature ofabout 17 mm (single curvature) or about 19 mm×12 mm (double curvature).In FIG. 10A the top is approximately 7 mm in diameter 1007. The outersurface of the implant may be approximately 1 mm thick or thicker 1009and may be about 70% porous, or greater. The hydrogel may be attached tothe top surface. The hydrogel in this example is a triple-networkhydrogel of BC-PVA-PAMPS and the BC is cemented to the porous top, whilethe PVA-PAMPS is impregnated into the BC. FIG. 10B shows a similarimplant to that shown in FIG. 10A, in which a hydrogel is attached(e.g., via cementing the nanofibrous portion of the hydrogel to theporous surface of the implant, as shown. The implant in FIG. 10B istitanium.

As mentioned above, any of these implant surfaces may include a porousstructure. The porosity of the implant surface may be, e.g., between 10%porous and 90% porous, e.g., between 30% porous and 90% porous, between55% porous and 95% porous, between 65% porous and 85% porous, etc.). Thedepth of the pores may also be varied. For example, the surface may beporous to a depth of between 0.1 mm and 5 mm, between 0.2 mm to 3 mm,between 0.5 mm to 2 mm (e.g., 0.2 mm or greater, 0.3 mm or greater, 0.5mm or greater, 0.75 mm or greater, 1 mm or greater, 1.5 mm or greater,etc.).

As mentioned, any appropriate nanofibrous network may be used,including, but not limited to nanofibrous bacterial cellulose. Othernanofibrous networks may include electrospun polymer nanofibers such aspoly(vinyl alcohol) (PVA) nanofibers, aramid nanofibers (e.g.,Aramid-PVA nanofibers), wet-spun silk protein nanofiber, chemicallycrosslinked cellulose nanofiber, or polycaprolactone fibers (e.g., 3Dwoven PCL fibers). In addition, any appropriate double network hydrogelsmay be used, including but not limited to PVA and PAMPS. For example,other hydrogel-forming polymers may include poly-(N,N′-dimethylacrylamide) (PDMAAm), copolymers of 1-vinylimidazole and methacrylicacid, double-network hydrogels based on amphiphilic triblock copolymers,polyampholyte hydrogels, a PVA-tannic acid hydrogel, a poly(N-acryloyl)glycinamide hydrogel, polyacrylic acid-acrylamide-C18 hydrogel,Guanine-boric acid reinforced PDMAAm, polyelectrolyte hydrogels, apoly(acrylonitrile-co-1-vinylimidazole) hydrogel (e.g., a mineralizedpoly(acrylonitrile-co-1-vinylimidazole) hydrogel), a polyacrylicacid-Fe3+-chitosan hydrogel, a poly(methacrylic acid) gel, a Grapheneoxide/Xonotlite reinforced polyacrylamide (PAAm) gel, a poly(stearylmethacrylate)-polyacrylic acid gel, an annealed PVA-polyacrylic acidhydrogel, supramolecular hydrogels from multiurea linkage segmentedcopolymers, polyacrylonitrile-PAAm hydrogel, a microsilica reinforcedDMA gel, a Agar-polyhydroxyethylmethacrylate gel, apolyfacryloyloethyltrimethylammonium chloride hydrogel, apoly(3-(methylacryloylamino)propyl-trimethylammonium chloride hydrogel,a poly(sodium p-styrenbesulfonate) hydrogel, a polyethylene glycoldiacrylate hydrogel, a polyethylene glycol hydrogel, or hydrogelscomposed of a combination of these polymers.

The implants described herein may be formed of any appropriate material,including, but not limited to titanium and stainless steel. For example,a hydrogel may be attached as described herein to an implant surface(e.g., base, including a porous base) that is formed of a stainlesssteel alloy, other titanium alloys, Co—Cr alloys, tantalum, gold,niobium, bone, Al oxide, Zr oxide, hydroxyapatite, tricalcium phosphate,calcium sodium phosphosilicate (Bio glass), poly(methyl methacrylate),polyether ether ketone, polyethylene, polyamide, polyurethane,polytetrafluoroethylene, or other materials used for making implants.

Any of the implants described herein may include a hydrogel having asurface that is substantial smooth and/or is shaped in a predeterminedconfiguration, such as (but not limited to) concave, convex,saddle-shaped, etc. For example, any of these apparatuses (e.g.,implants) may have a surface roughness that is less than 30 microns. Insome cases, the surface may be formed smooth by molding. In some cases,the surface may be formed smooth by polishing or sanding. For example,once the additional hydrogel components have formed the network (e.g.,the nanofibrous-reinforced network), the hydrogel coating may optionallybe finished by polishing; in particular, the surface may be sanded topolish to a roughness of less than 30 microns. Polishing may beperformed by sanding (e.g., using a fine grit sanding surface, such as a600, 400, 320, etc. grit).

FIG. 11 is a flowchart illustrating an exemplary method of making anapparatus as described herein. The surface to which the hydrogel is tobe attached, e.g., an implant surface, may optionally be prepared 1101.For example, the surface may be made porous. In some examples, theporosity may be at least 0.5 mm deep (e.g., 1 mm deep or deeper). Theporosity may be described as the percent porosity (e.g., between 10% and90% porous, between 20% and 90%, greater than 30%, greater than 40%,greater than 50%, etc.).

The nanofibrous portion may then be prepared for attachment to thesurface 1103. For example, the nanofibrous portion dried (e.g., freezedried). The nanofibrous portion may be applied dry or substantially dry,to the attachment surface 1105. The nanofibrous portion may then besecured to the surface 1107. In some variations an adhesive (e.g.,cement) may be applied to the surface before the nanofibrous portion isapplied and/or the adhesive may be applied onto the nanofibrous portionon the surface. In some variations the adhesive may be applied to thenanofibrous portion prior to attaching to the surface. In some examplesan adhesive is not used at all.

The adhesive, if used, may be applied to dry (e.g., for a predeterminedtime, e.g., 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 24 hours, etc.)at a drying temperature (e.g., room temperature, 30 degrees, etc.). Oncedried, the nanofibrous portion that is cemented to the surface mayoptionally be rehydrated 1109, e.g., by the addition of an aqueoussolution.

The nanofibrous portion may then be infiltrated by the other componentsof the hydrogel, which become impregnated into the nanofibrous portionsecured onto the surface 1111. The other components may include one ormore polymer components capable of forming a hydrogel and that arecrystallizable upon a subsequent annealing process. In some examples,the polymer components include polyvinyl alcohol (PVA). In someexamples, the polymer component only includes PVA.

Once the polymer hydrogel component is infused within the nanofibrousportion, a mechanical strengthening process may be implemented tostrengthen the hydrogel. The mechanical strengthening process mayinclude drying, annealing and rehydrating the hydrogel. Drying and/orannealing may include heating the hydrogel to a predeterminedtemperature (e.g., ranging from 90° C. to 140° C.), followed byrehydration (e.g., in PBS solution). The resulting hydrogel may have anincreased crystalline structure. For example, the interstitial polymerhydrogel (e.g., PVA) may have a crystallinity of at least 20%. In someexamples, the tensile strength of the resulting hydrogel is at least 40MPa. In some examples, the compressive strength of the resultinghydrogel is at least 59 MPa. In addition, the hydrogel may have a watercontent of about 20 wt % or greater. Once the mechanical strengtheningprocess is complete, the hydrogel surface may optionally be polished1115.

In some examples the apparatuses described herein may form part of asurgical implant for treating a defect, such as an osteochondral defect.For example, a surgical implant may include a surface that is covered ina hydrogel; this surface may act an interface between one or more otherbody regions, including hard tissues, such as bone and cartilage. Repairof a cartilage lesion with a hydrogel may benefit from long-termfixation of the hydrogel in the defect site. Attachment of a hydrogel toa base (substrate) that allows for integration with bone could enablelong-term fixation of the hydrogel, but current methods of forming bondsto hydrogels have less than a tenth of the shear strength of theosteochondral junction. The apparatuses and methods described herein mayinclude bonding a hydrogel to a surface (e.g., base) with a shearstrength that is many times larger than has been previously achieved.

FIGS. 12A-12C illustrate a brief overview of an example of how thehydrogel may be attached to a metal base (e.g., of the top bearingsurface). In this example freeze-dried BC sheets were cut into octagonalshapes with 8 projections (e.g., “legs”) that can be bent over the edgesof the implant, as shown in the example of FIG. 12D. This cut may removeexcess BC that would otherwise be folded up on the sides of thecylinder. The pieces of cut BC were then placed into a fixture thatfacilitates centering and alignment of the ring clamp with the pieces ofBC and the metal rod. The metal rod was pushed down through the fixtureso that the ring pushed the pieces of BC onto the metal rod. Thisprocess of pushing the ring over the BC and onto the rod could also bedone by hand. The use of an alignment features, such as shown in FIGS.12B-12C may help consistently center the pieces during assembly. Thesample may then be clamped, e.g., by heated in an oven at 90° C. toinitiate clamping in a shape-memory alloy material preset as describedherein (which starts at a temperature of 50° C.). The part was thenheated in a hydrothermal bomb at 120° C. for 24 hours with PVA toinfiltrate the polymer into the BC. The part was then dried, annealed,and rehydrated as described herein.

The following are example methods for preparing and testing varioushydrogel samples described herein.

Example 1: Fabrication of BC-PVA-PAMPS Hydrogel

BC sheets were pressed to be 0.5 mm thick and placed into a hydrothermalreactor with a mixture of polyvinyl alcohol (PVA) (40 wt. %) anddeionized water (60 wt. %). The hydrothermal reactor was sealed andheated at 120° C. for 24 hours to allow the PVA to diffuse into thevoids of BC and form a BC-PVA hydrogel. The BC-PVA hydrogel was removedfrom the reactor when hot (e.g., greater than 85° C.). The residual PVAsolution was removed by scrapping the surface of the BC-PVA samples witha metal spatula. The samples were frozen at −78° C. for 30 minutes andthawed at room temperature to physically crosslink the PVA network. TheBC-PVA hydrogel was then soaked in a solution of2-acrylamido-2-methylpropanesulfonic acid sodium salt (AMPS) (30 wt. %),N,N′-methylenediacrylamide (MBAA) (60 mM),2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (12959) (50 mM) andpotassium persulfate (KPS) (0.5 mg mL-1) for 24 hours. The hydrogel wascured with an ultraviolet (UV) transilluminator for 15 minutes on eachside, and further cured in an oven at 60° C. for 8 hours to ensure evenand complete curing. The resulting BC-PVA-PAMPS hydrogel was stored inphosphate buffered saline (PBS) for at least 24 hours before furthercharacterization.

Example 2: Fabrication of Annealed BC

BC sheets were pressed to be 0.5 mm thick. The BC sheets were thenplaced into a 90° C. oven for 24 hours before being annealed at 90° C.for an additional hour. The resulting annealed BC was cut into thedesired shape and stored in 0.15 M PBS for at least 24 hours.

Example 3: Fabrication of PVA Hydrogel

To fabricate the PVA hydrogel, a slurry of PVA (40 wt. %) and DI water(60 wt. %) were mixed in a metal baking pan (diameter: 203.2 mm) andheated at 120° C. for 20 minutes in an autoclave sterilizer. To makeannealed PVA hydrogel, the resulting hydrogel was dried in an oven at90° C. for 24 hours before being annealed at 90° C., 120° C. or 140° C.for an additional hour. To make freeze-thawed PVA hydrogel, theautoclaved hydrogel was frozen at −80° C. for 30 minutes and thawed at23° C. for 30 minutes. The resulting PVA hydrogel was cut into thedesired shape and stored in 0.15 M PBS for at least 24 hours beforetests.

Example 4: Fabrication of Annealed BC-40 wt. % PVA Hydrogel

BC sheets were pressed to be 0.5 mm thick and placed into a hydrothermalreactor with a mixture of PVA (40 wt. %) and DI water (60 wt. %). Thehydrothermal reactor was sealed and heated at 120° C. for 24 hours toallow the PVA to diffuse into the voids of BC and form a BC-PVAhydrogel. The BC-PVA hydrogel was removed from the reactor when hot(e.g., greater than 85° C.). Note the hydrothermal reactor waspressurized with hot steam and created a burn hazard, so personalprotective equipment including lab coat, heat resistant gloves andfull-coverage face shields should be used when opening the reactor. Theresidual PVA solution was removed by scrapping the surface of the BC-PVAsamples with a metal spatula. The samples were dried in an oven at 90°C. for 24 hours before annealing at 90° C., 120° C. or 140° C. for anadditional hour. The resulting annealed BC-PVA hydrogel was cut into adesired shape and stored in 0.15 M PBS for at least 24 hours beforetests.

Example 5: Fabrication of Annealed BC-10 wt. % PVA Hydrogel

BC sheets were pressed to be 0.5 mm thick and placed into a baking pan(15.6 cm×8.6 cm×4.2 cm). Approximately 30 mL of 10 wt. % PVA solutionwas added to the baking pan. The baking pan was placed in an oven at 90°C. for 24 hours and annealed at 90° C. for an additional hour. Theresulting annealed BC-PVA hydrogel was cut into the desired shape andstored in 0.15 M PBS for at least 24 hours.

Example 6: Fabrication of Annealed BC-PVA-PAMPS Hydrogel

BC sheets were pressed to be 0.5 mm thick and placed into a hydrothermalreactor with a mixture of PVA (40 wt. %) and DI water (60 wt. %). Thehydrothermal reactor was sealed and heated at 120° C. for 24 hours toallow the PVA to diffuse into the voids of BC and form a BC-PVAhydrogel. The BC-PVA hydrogel was removed from the reactor when hot(>85° C.). Note the hydrothermal reactor was pressurized with hot steamand created a burn hazard, so personal protective equipment including alab coat, heat resistant gloves and full-coverage face shields should beused when opening the reactor. The residual PVA solution was removed byscrapping the surface of the BC-PVA samples with a metal spatula. Thesamples were dried in an oven at 90° C. for 24 hours before beingannealing at 90° C., 120° C. or 140° C. for an additional hour. Theannealed BC-PVA hydrogel was then soaked in a solution of AMPS (30 wt.%), MBAA (60 mM), 12959 (50 mM) and KPS (0.5 mg mL-1) for 24 hours. Thehydrogel was cured with a UV transilluminator (VWR International) for 15minutes on each side, and further cured in an oven at 60° C. for 8 hoursto ensure even and complete curing. The resulting annealed BC-PVA-PAMPShydrogel was stored in PBS for at least 24 hours before furthercharacterization.

Example 7: Fabrication of Hydrogel on the Stainless-Steel Pin

Preparation of all hydrogel samples started with cutting thefreeze-dried BC. The BC is cut in the shape of an octagon with diameterD and 8 legs which has leg length of L and widths of W=0.383D (See FIGS.2A-2D, for example). The sample was labeled as BC-D-L after cutting. The8-piece star shape (BC-D-L) was generated by MATLAB and loaded intoAdobe Illustrator. In Adobe Illustrator the stroke of the shape ischanged to 0.0001 pt to ensure accurate cutting. The file is sent to thelaser cutter (Epilog Fusion M2) by using the print function and thelaser cutter was selected as the printer. The vector process is used,with 100% speed, 20% power and 100% frequency. For cutting the BC, aclean metal plate was placed on the bed of the laser cutter, and thefreeze-dried BC was placed on onto the metal plate. Another metal platewas placed onto the edge of the BC to ensure the BC stays in place. Thefocus was adjusted, and the shape was cut by the machine. After cutting,the BC was collected and stored in a petri dish for future use.

For preparing the shear test samples, six pieces of BC were adhered tothe stainless-steel rod with one layer of cement and a clamp. Anoverview of the assembly method is shown in FIGS. 12A-12C and 13 . Astainless-steel test rod was machined to have a top section with adiameter of 5.2 mm and a height of 2 mm, and a bottom section with adiameter of 6.75 mm and a height of 13 mm. Three pieces of BC-6.5-2.25and 3 pieces of BC-6.5-2 were placed in an alignment fixture. As anoptional step an adhesive (e.g., Scotchbond Universal Adhesive) may beapplied to the layer of the BC in contact with the rod and the topsurface of the rod. If used, the adhesive was allowed to set for 20seconds before being blown by air for another 5 seconds. In one example,about 0.15 g of RelyX Ultimate Cement was then applied to the samesurfaces coated with the Scotchbond Universal Adhesive. The rod waspressed into the BC layers and then into a shape memory alloy ringclamp. The cement was cured for 1 h. The sample was heated in an oven at175° C. for 10 min to shrink the clamp. The sample was then soaked in DIwater for 1 hour in a centrifuge tube before future use. The sample withBC on top then went through the specific hydrogel fabrication process.

Compression test samples were fabricated using the stainless-steel rodand a clamp, but without cement. Wear test samples were fabricated witha stainless-steel rod 5.7 mm in diameter and 38 mm in height, 3 piecesof BC-6.5-2, and the shape memory alloy ring, but without cement.

Example 8: Monotonic Tensile and Compression Tests

Monotonic tensile tests were carried out on an Instron 1321 (Instron,Norwood, MA, USA) and a TestResources 830 (TestResources, Shakopee, MN,USA) load frame at a rate of 0.25 mm s⁻¹. The finished hydrogel was cutinto an ASTM D638-14 Type V shape with a titanium hollow punch fortesting (see FIG. 1 for examples). The dimensions of the samples weremeasured with a caliper before testing. The ultimate tensile strength(UTS) was the maximum stress measured before fracture in the case of theBC-PVA samples, or the maximum compressive stress at 80% strain for thePVA samples. The tensile modulus was taken as the slope of thestress-strain curve at a stress of 1 MPa for comparison with previousstudies of human cartilage.

The compressive properties of all samples were measured with an axialTorsion System (TestResources 830LE63). Cylindrical samples of PVA werecut out of films of hydrogel with a hollow steel punch with a diameterof 4 mm. BC-PVA samples were attached to a metal pin for compressiontesting in order to have a sample that was sufficiently thick. Thedimensions of the samples were measured with a caliper before testing.The compressive properties were measured with a strain rate of 0.05 s⁻¹.The ultimate compressive strength was taken as the maximum stressmeasured before fracture. The compressive modulus was derived as theslope of the stress-strain curve at a stress of 0.4 MPa for comparisonwith previous studies of human cartilage.

Example 9: Differential Scanning Calorimetry

Differential scanning calorimetry (DSC) was performed on hydrogels todetermine the crystallinity of the PVA. The tests were completed on a TATGA550. In a typical experiment, a hydrogel sample of approximately 5 mgwas placed in an aluminum pan under a nitrogen gas flow and heated at arate of 10° C./min from 25° C. to 300° C. Typical thermograms for PVA,BC-PVA and BC-PVA-PAMPS hydrogels are shown in FIGS. 4A-4D.

The calculation for how much of the PVA was crystallized, i.e., thedegree of crystallinity, was adopted from Hassan et al.22 After the DSCthermogram was acquired, the area under the melting peak over the range140-220° C. (as shown in FIGS. 5A and 5B) was integrated to obtain avalue with units of J·° C.·S-1·g-1. This number was then divided by theheating rate (0.17° C.·S⁻¹) to obtain ΔH (J·g⁻¹). The crystallinity ofPVA was then calculated by dividing ΔH for the sample by the heatrequired for melting a 100% crystalline PVA sample, ΔH_(e)=138.6 J/g,and the weight fraction of PVA in the sample, w_(PVA),

$X_{PVA} = \frac{\Delta H}{w_{PVA} \times \Delta H_{c}}$

where χ_(PVA) is the crystallinity of the PVA.

Example 10: Measurement of Solid Weight Fraction

The weight of approximately 1 g of hydrated hydrogel was measured beforedrying at 90° C. for 24 hours. The weight of the dehydrated sample wasthen measured. The weight after dehydration was divided by the weightbefore dehydration to determine the solid weight fraction of thehydrogel sample.

Example 11: Wear Testing

The wear resistance of the hydrogels and porcine cartilage samples weredetermined with the pin-on-disk setup shown in FIGS. 6A-6D. Thepin-on-disk method was used with an Anton Paar Rheometer (MR302) and atribology accessory (SCF7). Cartilage samples were harvested from pigfemurs with an osteochondral autograft transfer system (Arthrex). Thefemurs were purchased from a local grocery store and frozen at −78° C.before harvesting the samples. Hydrogel samples were polished with #600,#800, #1000, #1200, #1500, #2000, #2500 and #3000 sandpapers to makethem smooth prior to testing. A hydrogel pin was fabricated by using themethod described in Example 7 above. A disk of hydrogel or porcinecartilage with a diameter of 12.7 mm was adhered with cyanoacrylate glue(Gorilla Glue Company) to the sample holder. The testing parameters wereas follows: 1,000,000 rotations; angular speed: 319 rounds per minute(maximum linear velocity: 100 mm s-1); normal force: 28.26 N (pressure:1 MPa). A pressure of 1 MPa was applied to each sample for 5 minutesbefore tests started. The tests were performed in FBS. FBS is often usedduring wear tests to mimic the lubrication provided by synovial fluid.

After the wear test, the samples were rehydrated in FBS for 24 hours toallow the gels to recover from the applied pressure before the weardepth was measured with a High-Resolution X-ray Computed Tomography(Micro-CT) Scanner (Nikon XTH 225 ST). A 3D model of the reconstructedMicro-CT images was rendered with Avizo 9 Lite. To measure the weardepth, a slice of the 3D model was taken in the middle of the wear mark.The wear depths were measured from the images of the middle slices withImageJ.

For calculating the COF, we determined the total friction force (F) fromthe torque (T) and the radius of the pin in the pin-on-disk setup (R):

$F = \frac{3T}{2R}$

The COF can then be calculated by:

${COF} = \frac{F}{F_{N}}$

Here F_(N) is the normal force (28.26 N). The linear velocity (v) wascalculated by:

v=ωR

where ω was the angular speed of the pin.

The results described above are particularly and unexpectedlysurprising, showing that annealed BC-PVA as described herein provide amaterial that maintains a low coefficient of friction (0.21) over 1million or more cycles of wear that is similar to cartilage (0.2). Incontrast, annealed PVA by itself has a COF that increases from 0.033 to0.135 over the million cycles. It is particularly surprising that addingthe BC to the PVA resulted in a long-term COF that is 6.5 times lowerthan PVA by itself. This lower COF enabled by BC is critical topreventing wear of an opposing cartilage surface. Along the same lines,it is surprising that adding BC to annealed PVA decreased the wear depthby more than three times relative to annealed PVA by itself. Thisgreater wear resistance enabled by BC may also be critical for the longterm durability of the implant.

Example 12: Shear Testing

Shear testing was performed on an 830LE63 Axial Torsion Test Machineequipped with a 100 pound (lb) load cell. Each test was performed in acustomized shear test fixture (see FIG. 13 ). For shearing of cartilageor hydrogel on metal samples, the sample was secured in a cylindricalhole in the left side of the fixture. The hole size was 6 mm for the pigplug and 7 mm for the hydrogel samples. Spacers were added underneaththe samples to precisely align the shear plane to the cartilage-bone orhydrogel-metal interface. The right side of the fixture was machined tohave a complementary half-cylinder that was used to push the hydrogel orcartilage off of their substrates. The diameter of the righthalf-cylinder matched that of the left side (either 6 or 7 mm). Rubberwas placed between the sample and the right shear fixture to applypressure during the shear test in order to minimize cleavage andpeeling. A crosshead displacement rate of 2 mm min-1 was used for allthe measurements.

Supplemental Materials

As mentioned, in some of these examples, Bacterial Cellulose (BC) waspurchased from Gia Gia Nguyen Co. Ltd. Poly(vinyl alcohol) (PVA) (fullyhydrolyzed, molecular weight: 145,000 g mol⁻¹),N,N′-methylenediacrylamide (MBAA, 97.0%),2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (12959), potassiumpersulfate (KPS) and 2-acrylamido-2-methylpropanesulfonic acid sodiumsalt (AMPS, 50 wt. % solution in water) were purchased from SigmaAldrich. Phosphate buffered saline (PBS) was purchased from VWRInternational. Fetal bovine serum (FBS, Canada origin, collected fromcattle typically 12-24 months old) was purchased from Corning. Shapememory alloy ring clamps were purchased from Intrinsic Devices.

In some examples, BC sheets were pressed to be 0.5 mm thick and placedinto a hydrothermal reactor with a mixture of PVA (40 wt. %) and DIwater (60 wt. %). The hydrothermal reactor was sealed and heated at 135°C. for 24 hours to allow the PVA to diffuse into the voids of BC andform a BC-PVA hydrogel. The BC-PVA hydrogel was removed from the reactorwhen hot (>85° C.). Note the hydrothermal reactor was pressurized withhot steam and is a burn hazard, so personal protective equipmentincluding lab coat, heat resistant gloves and full-coverage face shieldsshould be used when opening the reactor. The residual PVA solution wasremoved by scrapping the surface of the BC-PVA samples with a metalspatula. The samples were frozen at −78° C. for 30 minutes and thawed atroom temperature to physically crosslink the PVA network. The BC-PVAhydrogel was then soaked in a solution of AMPS (30 wt. %), MBAA (60 mM),12959 (50 mM) and KPS (0.5 mg mL-1) for 24 hours. The hydrogel was curedwith a UV transilluminator (VWR International) for 15 minutes on eachside, and further cured in an oven at 60° C. for 8 hours to ensure evenand complete curing. The resulting BC-PVA-PAMPS hydrogel was stored inPBS for at least 24 hours before further characterization.

As mentioned, BC sheets were pressed, e.g., to be about 0.5 mm thick. Insome examples, the BC sheets were then placed into a 90° C. oven for 24hours before being annealed at 90° C. for an additional hour. Theresulting annealed BC was cut into the desired shape and stored in PBS(0.15 M) for at least 24 hours.

Any appropriate method may be used to fabricate the PVA hydrogel. Forexample, to fabricate a PVA hydrogel, a slurry of PVA (40 wt. %) and DIwater (60 wt. %) were mixed in a metal baking pan (diameter: 203.2 mm)and heated at 120° C. for 20 minutes in an autoclave sterilizer. To makeannealed PVA hydrogel, the resulting hydrogel was dried in an oven at90° C. for 24 hours before being annealed at 90° C., 120° C. or 140° C.for an additional hour. To make freeze-thawed PVA hydrogel, theautoclaved hydrogel was frozen at −80° C. for 30 minutes and thawed at23° C. for 30 minutes. The resulting PVA hydrogel was cut into thedesired shape and stored in PBS (0.15 M) for at least 24 hours beforetesting.

Annealed BC-40 wt. % PVA Hydrogel may be fabricated in any appropriatemanner. For example, BC sheets may be pressed to be 0.5 mm thick andplaced into a hydrothermal reactor with a mixture of PVA (40 wt. %) andDI water (60 wt. %). The hydrothermal reactor was sealed and heated at135° C. for 24 hours to allow the PVA to diffuse into the voids of BCand form a BC-PVA hydrogel. The BC-PVA hydrogel was removed from thereactor when hot (>85° C.). The residual PVA solution was removed byscrapping the surface of the BC-PVA samples with a metal spatula. Thesamples were dried in an oven at 90° C. for 24 hours before annealing at90° C., 120° C. or 140° C. for an additional hour. The resultingannealed BC-PVA hydrogel was cut into a desired shape and stored in PBS(0.15 M) for at least 24 hours before tests.

In some examples Annealed BC-10 wt. % PVA Hydrogel was fabricated bypressing BC sheets to be about 0.5 mm thick and placed into a baking pan(15.6 cm×8.6 cm×4.2 cm). Approximately 30 mL of 10 wt. % PVA solutionwas added to the baking pan. The baking pan was placed in an oven at 90°C. for 24 hours and annealed at 90° C. for an additional hour. Theresulting annealed BC-PVA hydrogel was cut into the desired shape andstored in PBS (0.15 M) for at least 24 hours.

Annealed BC-PVA-PAMPS Hydrogel was fabricated by pressing BC sheets tobe about 0.5 mm thick and placed into a hydrothermal reactor with amixture of PVA (40 wt. %) and DI water (60 wt. %). The hydrothermalreactor was sealed and heated at 120° C. for 24 hours to allow the PVAto diffuse into the voids of BC and form a BC-PVA hydrogel. The BC-PVAhydrogel was removed from the reactor when hot (>85° C.). Note thehydrothermal reactor was pressurized with hot steam and created a burnhazard, so personal protective equipment including a lab coat, heatresistant gloves and full-coverage face shields should be used whenopening the reactor. The residual PVA solution was removed by scrappingthe surface of the BC-PVA samples with a metal spatula. The samples weredried in an oven at 90° C. for 24 hours before being annealing at 90°C., 120° C. or 140° C. for an additional hour. The annealed BC-PVAhydrogel was then soaked in a solution of AMPS (30 wt. %), MBAA (60 mM),12959 (50 mM) and KPS (0.5 mg mL-1) for 24 hours. The hydrogel was curedwith a UV transilluminator (VWR International) for 15 minutes on eachside, and further cured in an oven at 60° C. for 8 hours to ensure evenand complete curing. The resulting annealed BC-PVA-PAMPS hydrogel wasstored in PBS (0.15 M) for at least 24 hours before furthercharacterization.

Fabrication of Hydrogel on a Stainless-Steel Pin

In some examples, Preparation of hydrogel samples on a stainless-steelpin started with cutting the freeze-dried BC. The BC was cut in theshape of an octagon with diameter D, 8 legs of length of L, and widthsof W=0.383D. The sample was labeled as BC-D-L after cutting. The 8-piecestar shape (BC-D-L) was generated by MATLAB and loaded into AdobeIllustrator. In Adobe Illustrator the stroke of the shape was changed to0.0001 pt to ensure accurate cutting. The file was sent to the lasercutter (Epilog Fusion M2) using the print function and the laser cutterwas selected as the printer. The vector process was used, with 100%speed, 20% power and 100% Frequency. For cutting the BC, a clean metalplate was placed on the bed of the laser cutter, and the freeze-dried BCwas placed onto the metal plate. Another metal plate was placed onto theedge of the BC to ensure the BC did not move. The focus was adjusted,and the shape was cut by the machine. After cutting, the BC wascollected and stored in a petri dish for future use.

For preparing the shear test samples, six pieces of BC were adhered tothe stainless-steel rod with one layer of cement and a clamp. Astainless-steel test rod was machined to have a top section with adiameter of 5.2 mm and a height of 2 mm, and a bottom section with adiameter of 6.75 mm and a height of 13 mm. Three pieces of BC-6.5-2.25and 3 pieces of BC-6.5-2 were placed in an alignment fixture. ScotchbondUniversal Adhesive was applied to the layer of the BC in contact withthe rod and the top surface of the rod. The adhesive was allowed to setfor 20 seconds before being blown by air for another 5 seconds. About0.15 g of RelyX Ultimate Cement was then applied to the same surfacescoated with the Scotchbond Universal Adhesive. The rod was pressed intothe BC layers and then into a shape memory alloy ring clamp. The cementwas cured for 1 h. The sample was heated in an oven at 175° C. for 10min to shrink the clamp. The sample was then soaked in DI water for 1hour in a centrifuge tube before future use. The sample with BC on topthen went through the specific hydrogel fabrication process.

Compression test samples were fabricated using the stainless-steel rodand a clamp, but without cement. Wear test samples were fabricated witha stainless-steel rod 5.7 mm in diameter and 38 mm in height, 3 piecesof BC-6.5-2, and the shape memory alloy ring, but without cement.

Monotonic tensile tests were carried out on an Instron 1321 load frame(Instron, Norwood, MA, USA) and a Test Resources 830LE63 Axial TorsionTest Machine (TestResources, Shakopee, MN, USA) at a rate of 0.25 mms⁻¹. The finished hydrogel was cut into an ASTM D638-14 Type V shapewith a titanium hollow punch for testing (see FIGS. 15A-15D, forexamples). The dimensions of the samples were measured with a caliperbefore testing. The ultimate tensile strength (UTS) was the maximumstress measured before fracture in the case of the BC-PVA samples, orthe maximum compressive stress at 80% strain for the PVA samples. Thetensile modulus was taken as the slope of the stress-strain curve at astress of 1 MPa for comparison with previous studies of human cartilage.

The compressive properties of all samples were measured with a TestResources 830LE63 Axial Torsion Test Machine. Cylindrical samples of PVAwere cut out of films of hydrogel samples with a hollow steel punch witha diameter of 4 mm. BC-PVA samples were attached to a metal pin forcompression testing in order to have a sample that was sufficientlythick. The dimensions of the samples were measured with a caliper beforetesting. The compressive properties were measured with a strain rate of0.05 s⁻¹. The compressive strength was taken stress at a strain of 0.8,or the stress at fracture if the material failed before a strain of 0.8.The compressive modulus was derived as the slope of the stress-straincurve at a stress of 0.4 MPa for comparison with previous studies ofhuman cartilage.

Differential scanning calorimetry (DSC) was performed on hydrogelsamples to determine the crystallinity of the PVA. The tests werecompleted on a TA Instruments TGA550. In a typical test, a hydrogelsample of approximately 5 mg was placed in an aluminum pan and heated ata scanning rate of 10° C./min under a nitrogen gas flow from 25° C. to300° C. Typical thermograms for PVA, BC-PVA and BC-PVA-PAMPS hydrogelsare shown in FIGS. 17A-17C.

The calculation for how much of the PVA was crystallized, i.e., thedegree of crystallinity, was performed. After the DSC thermogram wasacquired, the area under the melting peak over the range 140-220° C. (asshown in FIG. 18 ) was integrated to obtain a value with units of J·°C.·S⁻¹·g⁻¹. This number was then divided by the heating rate (0.17°C.·S⁻¹) to obtain ΔH (J·g⁻¹). The crystallinity of PVA (X_(PVA)), wasthen calculated by dividing ΔH for the sample by the heat required formelting a 100% crystalline PVA sample, (ΔHc=138.6 J/g) and the weightfraction of PVA in the sample, w_(PVA):

$X_{PVA} = \frac{\Delta H}{w_{PVA} \times \Delta H_{c}}$

The weight of approximately 1 g of hydrated hydrogel was measured beforedrying at for 24 hours. The weight of the dehydrated sample was thenmeasured. The weight after dehydration was divided by the weight beforedehydration to determine the solid weight fraction of the hydrogelsample.

Fourier Transform Infrared (FTIR) spectroscopy was performed on hydrogelsamples to analyze changes in bonding after annealing. Hydrogel sampleswere cut into a 1 cm by 1 cm square before testing. The tests werecompleted on a Thermo Scientific Nicolet iS50 FT-IR. In a typical test,the sample was held under the detector with the number of scans set to32, resolution set to 4 (0.482 cm⁻¹) and format set to % transmittance.Typical FTIR spectra are shown in FIGS. 16A-16B.

The wear resistance of the hydrogels and porcine cartilage samples weredetermined with the pin-on-disk setup. The pin-on-disk method was usedwith an Anton Paar Rheometer (MR302) and a tribology accessory (SCF7).Cartilage samples were harvested from pig femurs with an osteochondralautograft transfer system (Arthrex). The femurs were purchased from alocal grocery store and frozen at −78° C. before harvesting the samples.Hydrogel samples were polished with #600, #800, #1000, #1200, #1500,#2000, #2500 and #3000 sandpapers to make them smooth prior to testing.A hydrogel pin was fabricated by using the method described in section2.8. A disk of hydrogel or porcine cartilage with a diameter of 12.7 mmwas adhered with cyanoacrylate glue (Gorilla Glue Company) to the sampleholder. The testing parameters were as follows: 1,000,000 rotations;angular speed: 319 rounds per minute (maximum linear velocity: 100 mms-1); normal force: 28.26 N (pressure: 1 MPa). A pressure of 1 MPa wasapplied to each sample for 5 minutes before starting the test. The testswere performed in FBS. FBS is often used during wear tests to mimic thelubrication provided by synovial fluid.

After the wear test, the samples were rehydrated in FBS for 24 hours toallow the gels to recover from the applied pressure before the weardepth was measured with a High-Resolution X-ray Computed Tomography(Micro-CT) Scanner (Nikon XTH 225 ST). A 3D model of the reconstructedMicro-CT images was rendered with Avizo 9 Lite. To measure the weardepth, a slice of the 3D model was taken in the middle of the wear mark.The wear depths were measured from the images of the middle slices withImageJ.

For calculating the COF, we determined the total friction force (F) fromthe torque (7) and the radius of the pin in the pin-on-disk setup (R):

$F = \frac{3T}{2R}$

The COF can then be calculated by:

${COF} = \frac{F}{F_{N}}$

Here F N is the normal force (28.26 N). The linear velocity (v) wascalculated by

v=ωR

where ω was the angular speed of the pin.

Shear testing was performed on a Test Resources 830LE63 Axial TorsionTest Machine equipped with a 100 lb. load cell. Each test was performedin a customized shear test fixture (see FIG. 13 ). For shearing ofcartilage off bone or hydrogel off metal samples, the sample was securedin a cylindrical hole in the left side of the fixture. Spacers wereadded underneath the samples to precisely align the shear plane to thecartilage-bone or hydrogel-metal interface. The right side of thefixture was machined to have a complementary half-cylinder that was usedto push the hydrogel or cartilage off of their substrates. A rubberspacer was placed between the sample and the right shear fixture toapply pressure during the shear test in order to minimize cleavage andpeeling. A crosshead displacement rate of 2 mm min-1 was used for allthe measurements.

A human-sized osteochondral implant 20 mm in diameter was fabricated.The top surface of the implant had a radius of curvature of 20 mm tomatch the native curvature of the femoral condyle. This implant wasfabricated with 5 BC layers. A 0.25-mm-thick coating of commerciallypure titanium was applied to the stem of the implant and underneath thebase with a plasma spray process in order to improve integration withbone.

Tensile Compres- Compres- Tensile Modu- sive sive Strength lus StrengthModulus Composition (MPa) (MPa) (MPa) (MPa) Freeze-thawed BC-PVA 11.1115.3 55.3 15.0 Annealed BC-PVA 50.5 503.9 98.1 9.57 AnnealedBC-PVA-PAMPS 22.1 179.0 60.4 16.5 Annealed PVA 15.6 24.5 56.7 12.1Freeze-thawed PVA 0.26 <0.14 14.8 2.41 Human cartilage 8.1-40 58-22814-59 8.1-20.1 BC-PVA-PAMPS 22.6 227 23.0 15.2 CNC-PA-PAAm 16.5 232.431.1 65.4 BC-PAAm 40 114 5.1 10 Polyaramid nanofiber-PVA 5 9.1 4 26.5BC-gelatin 3.8 21 5.3 2.9 PVA-Agarose 14.6 6.38 3.66 0.09 PVA-HA/HAAC3.05 0.7 40.15 0.88 3D printed PCL scaffold-PVA 4.41 9.53 3 1.2 PVA-CS4.02 2.07 18 1.2

The table above shows mechanical properties of annealed BC-PVA, annealedBC-PVA-PAMPS, annealed PVA hydrogels, cartilage, and previously reportedhydrogels. This table compares the properties of the anneal BC-PVA tofreeze-thawed and other previous hydrogels showing a remarkableimprovement in properties.

Abbreviations used in this table: BC: bacterial cellulose; PVA:Poly(vinyl alcohol); PAMPS: poly(2-acrylamido-2-methyl-1-propanesulfonicacid sodium salt); PAAm: polyacrylamide; CNC: cellulose nanocrystal, PA:phenyl acrylate; HA: hydroxyapatite, HACC: 2-hydroxypropyltrimethylammonium chloride chitosan, PCL: polycaprolactone, CS: chitosan. For thesake of clarity, the references in this table were limited topublications that report all four metrics, i.e., strength and modulus intension and compression, and had a tensile and compressive strengthhigher than 3 MPa.

Any of the methods (including user interfaces) described herein may beimplemented as software, hardware or firmware, and may be described as anon-transitory computer-readable storage medium storing a set ofinstructions capable of being executed by a processor (e.g., computer,tablet, smartphone, etc.), that when executed by the processor causesthe processor to control perform any of the steps, including but notlimited to: displaying, communicating with the user, analyzing,modifying parameters (including timing, frequency, intensity, etc.),determining, alerting, or the like.

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the inventive subject matter disclosed herein and may be used toachieve the benefits described herein.

When a feature or element is herein referred to as being “on” anotherfeature or element, it can be directly on the other feature or elementor intervening features and/or elements may also be present. Incontrast, when a feature or element is referred to as being “directlyon” another feature or element, there are no intervening features orelements present. It will also be understood that, when a feature orelement is referred to as being “connected”, “attached” or “coupled” toanother feature or element, it can be directly connected, attached orcoupled to the other feature or element or intervening features orelements may be present. In contrast, when a feature or element isreferred to as being “directly connected”, “directly attached” or“directly coupled” to another feature or element, there are nointervening features or elements present. Although described or shownwith respect to one embodiment, the features and elements so describedor shown can apply to other embodiments. It will also be appreciated bythose of skill in the art that references to a structure or feature thatis disposed “adjacent” another feature may have portions that overlap orunderlie the adjacent feature.

Terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.For example, as used herein, the singular forms “a”, “an” and “the” areintended to include the plural forms as well, unless the context clearlyindicates otherwise. It will be further understood that the terms“comprises” and/or “comprising,” when used in this specification,specify the presence of stated features, steps, operations, elements,and/or components, but do not preclude the presence or addition of oneor more other features, steps, operations, elements, components, and/orgroups thereof. As used herein, the term “and/or” includes any and allcombinations of one or more of the associated listed items and may beabbreviated as “/”.

Spatially relative terms, such as “under”, “below”, “lower”, “over”,“upper” and the like, may be used herein for ease of description todescribe one element or feature's relationship to another element(s) orfeature(s) as illustrated in the figures. It will be understood that thespatially relative terms are intended to encompass differentorientations of the device in use or operation in addition to theorientation depicted in the figures. For example, if a device in thefigures is inverted, elements described as “under” or “beneath” otherelements or features would then be oriented “over” the other elements orfeatures. Thus, the exemplary term “under” can encompass both anorientation of over and under. The device may be otherwise oriented(rotated 90 degrees or at other orientations) and the spatially relativedescriptors used herein interpreted accordingly. Similarly, the terms“upwardly”, “downwardly”, “vertical”, “horizontal” and the like are usedherein for the purpose of explanation only unless specifically indicatedotherwise.

Although the terms “first” and “second” may be used herein to describevarious features/elements (including steps), these features/elementsshould not be limited by these terms, unless the context indicatesotherwise. These terms may be used to distinguish one feature/elementfrom another feature/element. Thus, a first feature/element discussedbelow could be termed a second feature/element, and similarly, a secondfeature/element discussed below could be termed a first feature/elementwithout departing from the teachings of the present invention.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising” means various components can be co-jointlyemployed in the methods and articles (e.g., compositions and apparatusesincluding device and methods). For example, the term “comprising” willbe understood to imply the inclusion of any stated elements or steps butnot the exclusion of any other elements or steps.

In general, any of the apparatuses and methods described herein shouldbe understood to be inclusive, but all or a sub-set of the componentsand/or steps may alternatively be exclusive, and may be expressed as“consisting of” or alternatively “consisting essentially of” the variouscomponents, steps, sub-components or sub-steps.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “about” or “approximately,” even if theterm does not expressly appear. The phrase “about” or “approximately”may be used when describing magnitude and/or position to indicate thatthe value and/or position described is within a reasonable expectedrange of values and/or positions. For example, a numeric value may havea value that is +/−0.1% of the stated value (or range of values), +/−1%of the stated value (or range of values), +/−2% of the stated value (orrange of values), +/−5% of the stated value (or range of values), +/−10%of the stated value (or range of values), etc. Any numerical valuesgiven herein should also be understood to include about or approximatelythat value, unless the context indicates otherwise. For example, if thevalue “10” is disclosed, then “about 10” is also disclosed. Anynumerical range recited herein is intended to include all sub-rangessubsumed therein. It is also understood that when a value is disclosedthat “less than or equal to” the value, “greater than or equal to thevalue” and possible ranges between values are also disclosed, asappropriately understood by the skilled artisan. For example, if thevalue “X” is disclosed the “less than or equal to X” as well as “greaterthan or equal to X” (e.g., where X is a numerical value) is alsodisclosed. It is also understood that the throughout the application,data is provided in a number of different formats, and that this data,represents endpoints and starting points, and ranges for any combinationof the data points. For example, if a particular data point “10” and aparticular data point “15” are disclosed, it is understood that greaterthan, greater than or equal to, less than, less than or equal to, andequal to 10 and 15 are considered disclosed as well as between 10 and15. It is also understood that each unit between two particular unitsare also disclosed. For example, if 10 and 15 are disclosed, then 11,12, 13, and 14 are also disclosed.

Although various illustrative embodiments are described above, any of anumber of changes may be made to various embodiments without departingfrom the scope of the invention as described by the claims. For example,the order in which various described method steps are performed mayoften be changed in alternative embodiments, and in other alternativeembodiments one or more method steps may be skipped altogether. Optionalfeatures of various device and system embodiments may be included insome embodiments and not in others. Therefore, the foregoing descriptionis provided primarily for exemplary purposes and should not beinterpreted to limit the scope of the invention as it is set forth inthe claims.

The examples and illustrations included herein show, by way ofillustration and not of limitation, specific embodiments in which thesubject matter may be practiced. As mentioned, other embodiments may beutilized and derived there from, such that structural and logicalsubstitutions and changes may be made without departing from the scopeof this disclosure. Such embodiments of the inventive subject matter maybe referred to herein individually or collectively by the term“invention” merely for convenience and without intending to voluntarilylimit the scope of this application to any single invention or inventiveconcept, if more than one is, in fact, disclosed. Thus, althoughspecific embodiments have been illustrated and described herein, anyarrangement calculated to achieve the same purpose may be substitutedfor the specific embodiments shown. This disclosure is intended to coverany and all adaptations or variations of various embodiments.Combinations of the above embodiments, and other embodiments notspecifically described herein, will be apparent to those of skill in theart upon reviewing the above description.

What is claimed is:
 1. An implant, comprising: an implant body having atop bearing surface; an anchoring base; and a cellulose-reinforcedhydrogel comprising: a cross-linked cellulose nanofiber network securedover the top bearing surface of the implant body; and an interstitialhydrogel portion within interstitial regions of the cross-linkedcellulose nanofiber network, wherein the interstitial hydrogel portionhas a crystallinity of 20% or greater.
 2. The implant of claim 1,wherein the interstitial hydrogel portion comprises polyvinyl alcohol(PVA).
 3. The implant of claim 1, wherein the cellulose-reinforcedhydrogel comprises at least 20% by weight of water.
 4. The implant ofclaim 1, wherein the cellulose-reinforced hydrogel has a tensilestrength exceeding 40 MPa.
 5. The implant of claim 1, wherein thecross-linked cellulose nanofiber network is chemically cross-linked. 6.The implant of claim 1, wherein the cross-linked cellulose nanofibernetwork comprises bacterial cellulose.
 7. The implant of claim 1,wherein the cellulose-reinforced hydrogel has a compressive strengthexceeding 59 MPa.
 8. The implant of claim 1, wherein the cross-linkedcellulose nanofiber network is secured over the top bearing surface by aclamp.
 9. The implant of claim 8, wherein the cross-linked cellulosenanofiber network comprises one or more sheets of bacterial cellulous(BC) held over the top bearing surface by a clamp secured to a lip orrim of the top bearing surface.
 10. An implant for a knee resurfacing,the implant comprising: a top bearing surface comprising acellulose-reinforced hydrogel comprising: a cellulose nanofiber network;and a hydrogel component impregnated in the cellulose nanofiber network,wherein the hydrogel component has a crystallinity of 20% or greater.11. The implant of claim 10, wherein the hydrogel component comprisespolyvinyl alcohol (PVA).
 12. The implant of claim 10, wherein thecellulose-reinforced hydrogel comprises at least 20% by weight of water.13. The implant of claim 10, wherein the cellulose-reinforced hydrogelhas a tensile strength exceeding 40 MPa.
 14. The implant of claim 10,wherein the cellulose-reinforced hydrogel is attached to a metallic baseof the top bearing surface with a shear strength exceeding 0.2 MPa. 15.The implant of claim 10, wherein the top bearing surface has acoefficient of friction (COF) that is not statistically greater thanthat of cartilage.